Geminivirus replicon, in planta directed evolution / selection system based thereon, and use

Abstract

A geminivirus replicon, an in planta directed evolution / selection system based thereon, and use. Provided is a geminivirus-based controllable artificial replicon, which comprises two LIRs derived from BeYDV; the two LIRs are located at two flanks of the artificial replicon, respectively. Also provided is an in planta directed evolution / selection system, which comprises the geminivirus-based controllable artificial replicon, a Rep and / or RepA protein, and a mutant or mutant library of a genetic element. The replication of the geminivirus-based controllable artificial replicon is configured to be associated with the desired function of the mutant of the genetic element. The provided in planta directed evolution / selection system is a universal, rapid, and efficient in planta directed evolution system, and can be used for generating new excellent alleles and assisting future agricultural breeding.

Description

Geminid replicons, their-based directed evolution / screening systems in plants, and their applications Technical Field

[0001] This invention belongs to the field of genetic engineering. Specifically, this invention relates to geminivirus replicons, plant-based directed evolution / screening systems, and their applications. Background Technology

[0002] Directed Evolution

[0003] Throughout their long history, organisms constantly undergo mutations. Some of these mutations are beneficial to survival, while others are detrimental. Under the pressure of natural selection, those beneficial mutations are preserved and accumulated, while those detrimental ones are eliminated—this process is evolution. For millions of years, organisms have adapted to their external environment through evolution.

[0004] However, this natural evolutionary process takes an extremely long time, often measured in millions of years; furthermore, the results of natural selection often do not match the traits or characteristics expected. Therefore, for half a century, mimicking the principles of natural selection and leveraging advancements in modern molecular biology, researchers have artificially constructed evolutionary systems in the laboratory. In these systems, numerous mutations are artificially created, and selective pressure is applied specifically according to desired functions and purposes to screen for genotypes with the desired characteristics. This molecular-level simulation of evolution is called directed evolution. Directed evolution allows for the modification of proteins even when the structural information and mechanism of action of the target protein are unknown. Therefore, directed evolution is one of the most effective methods for obtaining new functional proteins in contemporary molecular biology research.

[0005] A complete directed evolution system comprises three modules: diversification, selection, and iteration. Diversification refers to generating a wide variety of genotypes for the target gene through various pathways, which are then used for subsequent selection. Selection involves using meticulously designed gene pathways to screen various genotypes of the target gene, enriching those with the desired characteristics and eliminating those without. Iteration involves efficiently repeating the diversification and selection steps to obtain the target gene genotypes with the desired characteristics.

[0006] To date, dozens of directed evolution systems have been developed, achieving directed evolution in bacteria, yeast, animals, and plants, and obtaining the expected evolutionary products.

[0007] Directed Evolutionary Systems in Plants

[0008] Almost all reported directed evolution systems have been established in bacterial, yeast, and mammalian cell lines. This is because certain inherent factors in plants make it difficult to establish in vivo directed evolution systems. First, many directed evolution systems rely on high-frequency in vivo homologous recombination; however, in plants, homologous recombination efficiency is generally less than 0.1%. Second, while bacteria, yeast, and animal cells can efficiently prepare cell lines, only a very small number of plant species can currently produce such cell lines, and their reproducibility is low. Third, the lack of efficient transformation systems in plants makes high-throughput research in plants extremely labor-intensive and time-consuming. Finally, the operational throughput of plants is orders of magnitude lower than that of bacteria and yeast. These factors significantly hinder the development of in vivo directed evolution systems in plants, and substantial changes are unlikely in the short term.

[0009] However, developing plant-based directed evolution systems remains crucial. Firstly, plants, compared to other organisms, have different operating temperatures and pH levels. Most tools currently used in plants are derived from *E. coli* or mammals. *E. coli* and mammals generally thrive at 37°C, and the optimal operating temperature for proteins derived from them is also generally 37°C, while the optimal growth temperature for most plants is 20-25°C. Similarly, animal cells typically have a pH of 7.2-7.4, *E. coli* a pH of 7.0-7.5, while the pH of the plant cytoplasm is 5.6-5.9. These factors mean that proteins derived from *E. coli* and mammals, or products obtained through directed evolution systems dependent on *E. coli* and mammals, may not function as efficiently in plants due to thermodynamic and chemical factors. Secondly, plant cells possess unique anatomical structures. Compared to *E. coli*, plant cells, as eukaryotes, possess structures such as a nucleus and endoplasmic reticulum. Compared to animal cells, plant cells have structures like chloroplasts and cell walls. Proteins associated with these structures are difficult to evolve in *E. coli* or mammalian systems. Thirdly, plant cells possess unique cellular regulatory networks. Over the long course of evolution, complex regulatory networks have formed among various genetic elements within the cell, resulting in significant differences between prokaryotes and eukaryotes, and between plant and animal cells. Since no element is likely to function independently of this network, products obtained through directed evolution outside the plant cell system may not work effectively in a plant system. Furthermore, elements within the plant cell regulatory network are difficult to evolve in other biological systems. For these reasons, some elements that function efficiently in mammalian or *E. coli* systems do not exhibit similar activity in plant cells. Therefore, we aim to develop a plant cell-based directed evolution system.

[0010] In recent years, some studies have achieved preliminary results in directed evolution in plants. In 2019, Magdy M. Mahfouz first achieved directed evolution in rice using CRISPR-Cas (Butt et al., 2019). This work demonstrated the feasibility of directed evolution in plants for the first time.

[0011] Subsequently, Ren and Li et al. also conducted similar work in plants. Two studies utilized SpRY-CBE and Cas9-NG-ACBE base editors, respectively, to perform saturation mutations on the active regions of acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) in rice. They then screened for herbicide-resistant rice plants by applying bispyribac-sodium and fluroxypyr, thus completing the directed evolution of herbicide-targeting genes (Li et al., 2020; Ren et al., 2021).

[0012] Although these works have achieved directed evolution of target genes in plants, they are labor-intensive, time-consuming, and have low throughput (10). 2 Furthermore, the target genes are limited to compound target genes, making it difficult to extend to other target genes. Therefore, it is essential to establish a universal directed evolution system in plant systems.

[0013] Gemini virus

[0014] Geminiviruses are a class of uncoated, single-stranded circular DNA viruses. In agriculture, they can cause viral diseases and result in severe economic losses. They are named for their doublet-like appearance under an electron microscope. To date, over 520 species have been discovered within the Geminivirus family, and they are classified into 14 genera based on genomic structure, host range, and other characteristics, including Begomovirus, Mastrevirus, Curtovirus, Grablovirus, and Turncurtovirus. These viruses infect a wide variety of plants, including tomato, beet, rice, wheat, Arabidopsis, turnip, tobacco, and cucumber.

[0015] Most geminiviruses are monopodial viruses with a genome length of 2.3-2.8 kb, encoding 4-6 genes. Some geminiviruses in the genus *Aureobasidium* are dipodial viruses, with each component approximately 2.5-2.6 kb in length, encoding a total of 6-8 genes (Fiallo-Olive et al., 2021). Geminiviruses are generally transmitted by insects such as whiteflies, aphids, treehoppers, and leafhoppers, and generally cannot be transmitted from seed to seed.

[0016] Genomic structure of geminiviruses

[0017] The genome structures of different genera of geminiviruses are not entirely the same. The genera Begomovirus, Curtovirus, and Mastrevirus have the most members in the Geminivirus family.

[0018] The genus *Golden Bean Mosaic Virus* exists as monomorphs or multimorphs. Monomorphic viruses generally encode six genes: C1 (Rep), C2 (TrAP), C3 (REn), C4, V1 (CP), and V2 (MP). Some members of the genus *Golden Bean Mosaic Virus* also encode components such as C5 and V3. Among these, the C1 protein encodes the replication-associated protein (Rep). This protein is the most important encoded protein of geminiviruses, possessing DNA endonuclease and ligase activities, and possibly helicase activity, participating in the initiation and termination of geminivirus rolling circle replication. Rep is also a transcription factor that functions as an oligomer, binding to iterators on the viral LIR region (Bonnamy et al., 2023), participating in the regulation of viral genome expression, inhibiting the expression of complementary strand genes (C1–C4), and promoting the expression of viral strand genes (V1, V2). In addition, Rep can interact with endogenous genes such as retinoblastoma-related proteins (RBR), replication factor C (RFC), and replication protein A (RPA), thereby regulating the cell cycle, activating DNA synthesis genes in plant cells, and enabling viral DNA to replicate efficiently.

[0019] The structure of viruses in the *Saccharitovirus* genus is similar to that of single-dose *Bean Golden Mosaic Virus* genus. The genome structure of *Maize Stripe Virus* genus differs significantly from these two genera. Its viral strand also encodes MP and CP, but its complementary strand encodes only the Rep and RepA genes. The transcript of the Rep protein is composed of two exons spliced ​​together; it is homologous to C1 of *Bean Golden Mosaic Virus* genus and also participates in the initiation and termination of rolling circle replication. RepA is replication-associated protein A, sharing the N-terminus ~200 aa with the Rep protein. RepA lacks DNA endonuclease and ligase activity, but it possesses a conserved LXCXE motif, which can interact with phytoretinal glioma-associated proteins to regulate the plant cell cycle. Furthermore, RepA also exhibits transcriptional activator and gene silencing repressor activity.

[0020] In addition to coding genes, geminivirus genomes also contain non-coding regions. For members of the *Maize Stripe Virus* genus, their genomes have two non-coding regions, referred to as the long intergenic region (LIR) and the small intergenic region (SIR). The LIR functions similarly to the IR, while the SIR is a bidirectional transcription terminator and may be the binding site for endogenous primers during the viral transition from single-stranded circular DNA to double-stranded circular DNA.

[0021] Gemini replication mechanism

[0022] Geminiviruses replicate their genomes in plant cells through rolling circle replication (RCR) and recombination-dependent replication (RDR) (Bonnamy et al., 2023). RCR is the primary replication mode, while RDR likely plays only a secondary role.

[0023] After entering plant cells, the virus first transforms from ssDNA to dsDNA under the action of endogenous DNA or RNA primers. This process may involve endogenous primases and DNA polymerase α. Then, the viral complementary strand genes are expressed. During this process, the expression of genes such as Rep, RepA, and Ren regulates the host cell cycle. Rep then forms oligomers, specifically recognizing iterators on the viral LIR and clustering near the LIR. This process may cause a conformational change in nearby DNA, making it more susceptible to DNA replication initiation. Subsequently, Rep recognizes the conserved neck loop structure on the IR or LIR and exerts its DNA endonuclease activity, generating a single-strand break (nick) within a conserved 9 bp (typically 5'TAATATT / / AC3') of the neck loop structure. Next, Rep covalently binds to the 3' end of the DNA break and recruits endogenous DNA polymerase δ or polymerase ε to initiate rolling circle replication of the DNA. After the viral genome has completed DNA replication, Rep uses its DNA endonuclease and ligase activities to cut and ligate the newly formed DNA, ultimately creating a single-stranded circular DNA molecule, completing one replication cycle. Rolling circular replication is actually a very complex process involving many host factors, and many of its specific mechanisms are not yet fully understood.

[0024] Compared to RCR, RDR may be more of a supplement to DNA replication than the dominant mode.

[0025] Application of Geminid Replicons in Plant Biotechnology

[0026] A replicon is a DNA or RNA molecule capable of replicating within a cell. Due to the excellent DNA replication ability of geminiviruses in plant cells, they have been engineered into viral replicons and are widely used in plant biotechnology. Modification strategies for geminivirus replicons can be divided into two categories: full virus strategies and deconstructed virus strategies (Lozano-Duran, 2016). The full virus strategy involves inserting the target sequence into the geminivirus genome while maintaining the integrity of the viral genome. In this way, the virus can still achieve intercellular and systemic transmission within plants and express the target gene.

[0027] However, the whole-virus strategy has significant drawbacks. Geminiviruses have a low capacity to carry foreign fragments, with an upper limit of approximately 800 bp. On the one hand, the expression of each gene in a geminivirus is strictly regulated, and the insertion of foreign genes can lead to a decrease in viral infectivity. On the other hand, during migration, the size of geminiviruses is strictly limited by the mobile protein MP and plasmodesmata; the insertion of excessively long foreign genes can lead to a decrease in viral infectivity or recombination, resulting in the loss of foreign fragments.

[0028] To resolve this contradiction, researchers have developed a deconstruction virus strategy, which abandons the systemic movement of geminiviruses and retains only their ability to efficiently replicate in a rolling circle within cells. The encoding genes for MP, CP, and NSP in geminiviruses are not crucial for replication, so these are deleted, retaining only the replication proteins (C1–C4 in *Bean Golden Mosaic Virus* and *Beetroot Curly Top Virus*, and Rep / RepA in *Maize Stripe Virus*) and essential cistropic elements (IR, SIR, LIR). While this viral replicon cannot achieve intercellular or systemic transmission, its carrying capacity is greatly increased, generally enabling efficient replication of endogenous fragments up to 10 kb (Yu et al., 2020).

[0029] Thanks to this, the application of geminivirus replicons in plant biotechnology has been greatly expanded. Because geminiviruses can replicate efficiently in rolling circles within plant cells, their copy numbers can far exceed those of the nuclear genome. Furthermore, Rep and RepA can suppress gene silencing mechanisms in plants (Rodriguez-Negrete et al., 2013). Therefore, geminivirus vectors can achieve extremely high levels of exogenous gene expression, 40-100 times higher than conventional transformations (Mor et al., 2003). On the other hand, since plants do not contain biotoxins and are relatively inexpensive, geminivirus replicons can be used to efficiently produce products such as biological vaccines (Huang et al., 2009; Kim et al., 2015; Hager et al., 2022). However, the characteristics of geminiviruses mean they may interfere with plant growth, development, and regeneration, making it difficult to obtain stable transgenic plants containing a large number of geminivirus replicons. In 2013, Benjamin Dugdale et al. developed the In Plant Activation (INPACT) technology, which, using a plant ethanol induction system, obtained stable transformed plants containing geminivirus replicons. Only with the application of ethanol will the geminivirus initiate rolling circle replication and express the target protein in large quantities. This reduces the cost of expressing exogenous genes using geminiviruses.

[0030] In the field of gene editing, geminivirus replicons can be used as overexpression gene editing tools (such as ZFN, CRISPR-Cas, etc.) to achieve efficient gene editing in plants (Baltes et al., 2014; Butler et al., 2016; Yu et al., 2020). In addition, the high copy number of geminivirus vectors makes them suitable for carrying homologous recombination repair templates. The RepA, C3, and other genes encoded by geminiviruses can also adjust the cellular environment of plants, initiate the expression of genes required for homologous recombination, and make plant cells more susceptible to homologous recombination. Therefore, using replicons of the geminitroviruses Bean Yellow Dwarf Virus (BeYDV) and Wheat Dwarf Virus (WDV), researchers have achieved highly efficient homologous recombination in species such as rice, tomato, potato, and wheat (Butler et al., 2016; Cermak et al., 2017; Gil-Humanes et al., 2017; Wang et al., 2017; Dahan-Meir et al., 2018). Furthermore, using Wheat Dwarf Virus, Yifu Tian et al. developed a surrogate gene-editing system, improving the efficiency of gene editing in rice.

[0031] Broomviruses and Tomato Dwarf Viruses

[0032] Among plant viruses, those belonging to the Virgaviridae and Tombusviridae families cause severe crop diseases and yield losses every year. Developing resistance genes for these two types of viruses is of great significance in crop breeding.

[0033] NRC Network

[0034] In nature, paired nucleotide binding and leucine-rich repeat receptors (NLRs) intertwine with upstream sensing NLRs to form a gradient-structured network, known as the NLR network. In this network, helper NLRs connect multiple sensor NLRs within the same immune signaling pathway, or a single sensor NLR may correspond to multiple helper NLRs. Plant NLRs can be classified into three types based on their N-terminal signal transduction domains: TIR-NB-LRR (TNL), CC-NB-LRR (CNL), and CCR-NB-LRR (RNL). A common example is the NRC immune receptor network in Solanaceae plants. NRCs (NLRs - required for cell death) are a type of CNL that act as helper NLRs, mediating immune responses to different upstream sensor NLRs. In Nicotiana benthamiana, there are three NRCs: NRC2, NRC3, and NRC4 (Wu et al., 2017). In tomato, the NLR Prf, acting as a sensor NLR, forms an immune complex with its cofactor, the intracellular kinase protein Pto. This complex recognizes and binds to two type III effector proteins, AVRPto and AVRPtoB, of the bacterial pathogen *Pseudomonas syringae*, leading to cell death and conferring resistance to the pathogen to the plant. Studies have shown that co-expression of Pto and AVRPto in Nicotiana benthamiana, which contains the Prf homolog NbPrf, can activate an immune response that induces cell death. This response is dependent on NRC2 and NRC3 but not on NRC4. However, AvrPtoB only triggers Pto-mediated cell death when expressing SlPrf derived from tomato, and both NbPrf and SlPrf are NRC2 or NRC3-dependent sensor NLRs.Gpa2 is a potato-encoded CNL that confers resistance to two field populations of the potato cyst nematode (Globodera pallida). Studies have found that Gpa2 participates in the recognition of the Globodera pallida effector RBP1 (Gp-RBP1) through an initiation interaction protein or cofactor RanGAP2 (Ran GTPase Activating protein 2). RBP1 is a member of the SPRY (SP1a and Ryanodine receptor) family of cyst nematodes and is highly variable in nematode populations. Transient expression of Gpa2 and RBP1 in tobacco can induce cell death, and the cell death is dependent on downstream tobacco endogenous NRC2 or NRC3, but not on NRC4. In addition, the protein encoded by the gene Rx, which is closely related to Gpa2, as a sensor NLR, also requires the cofactor RanGAP2 to recognize the coat protein (CP) of potato virus X (PVX) to confer resistance to the virus to the plant and induce downstream NRC2 or NRC3-dependent cell death in tobacco.

[0035] Recent research shows that the N-terminus of tobacco NRC4 contains a conserved MADA motif in other NRCs, Arabidopsis-derived ZAR1, and some other monomeric NLRs. This motif degenerates in NRC-dependent sensor NLRs, suggesting that NRCs, ZAR1, and other monomeric NLRs may possess similar mechanisms of cell death induction. Recent studies have also found that Rx recognition of CP induces oligomerization in the downstream helper NLR NRC2, and that NRC2 oligomers accumulate on the cell membrane. Some helper NLRs, in their resting or active states, directly bind to phospholipids on the cell membrane via positively charged amino acid residues in the C / C domain, such as K84 of NRC4, to anchor themselves to the cell membrane, i.e., undergoing conformational changes upon activation.

[0036] SS15's ability to target and inhibit NRC

[0037] Plant-parasitic nematodes cause severe crop yield damage globally. Potato cyst nematodes (PCNs) affect approximately 9% of global potato production, resulting in stunted growth, yellowing leaves, and reduced yield. PCNs include the white nematode (Globodera pallida) and the gold nematode (Globodera rostochiensis), both of which are parasitic nematodes that infect only Solanaceae plants, including potatoes, tomatoes, and eggplants. SPRYSECs, a family of proteins containing the SPRY (SP1a and Ryanodine receptor) domain, are a class of proteins produced by the potato cyst nematode. These proteins are relatively ancient in evolution in animals, fungi, and plants, highly variable within nematode populations, and produced in the dorsal glandular cells of second-instar infected PCNs (J2s), functioning as effector proteins. The SPRY domain is considered crucial in protein-protein interactions. SPRY domain proteins with signal peptides (SPRYSECs) are expressed alone only in the early stages of PCN parasitism; however, these proteins without signal peptides are either not expressed or expressed throughout the entire life cycle. Several studies have shown that some SPRYSECs can inhibit ETI induced by plant resistance proteins. For example, SPRYSEC-19 interacts with CNL SW5F to suppress plant ETI. SPRYSEC member GpSPRY-414-2 inhibits ETI or cell death induced by the co-expression of the Gpa2 resistance gene and the non-toxic effector RBP-1 (RNA-binding protein-1), and reduces the reactive oxygen species (ROS) burst induced by flg22.

[0038] In recent years, Derevnina et al. (2021) screened and identified that AVRcaplb secreted by Phytophthora infestans and SPRYSEC15 (SS15) secreted by Globodera pallida both inhibit cell death caused by NRC autoactivation (Dong et al., 2021). SS15 is a member of the SPRYSECs family of secretory SPRY (SP1a and Ryanodine receptor) domain proteins unique to Globodera pallida (Ali et al., 2015). Recent studies have found that SS15 inhibits the oligomerization of NRC2 by directly linking the NB domain and CC structure of NRC2 to prevent intramolecular rearrangement, which further leads to the loss of protein function. By comparing the NRC2 single nucleotide polymorphism of NRC4 at the SS15 binding site, the study found that changing a single amino acid at the SS15-NRC2 interaction site enabled hepler NLR NRC2 to successfully evade the inhibition of SS15. This suggests that the artificial design based on structural analysis to de-target pathogens may be applied to future disease-resistant breeding. Summary of the Invention

[0039] The problem the invention aims to solve

[0040] To meet the ever-increasing demand for food, there is an urgent need for new methods that can efficiently create genetic variations to cultivate superior traits and aid in crop breeding. Directed evolution can endow target genes with stronger or more special properties in a short period of time. Although a number of directed evolution systems have been developed in microorganisms, they are not applicable to the evolution of genes for plant systems.

[0041] To address the aforementioned problems in existing technologies, this invention establishes a Geminivirus Replicon-Assisted in Planta Directed Evolution (GRAPE) system by developing an artificial replicon of a geminivirus and coupling the intended function of the target gene with the rolling circle replication of the replicon. GRAPE can complete 10 [unclear - possibly related to genetic engineering or genetic engineering] in a single tobacco leaf. 5 The system can screen for multiple types of targeted gene variants and complete a round of directed evolution within 4 days.

[0042] Using GRAPE, this invention has evolved a series of antiviral tRNAs that can efficiently read conserved stop codons in viruses, thereby conferring broad-spectrum viral resistance to plants. This is the first report of tRNAs being used for the control of plant viruses.

[0043] Furthermore, this invention utilizes GRAPE to evolve the tobacco endogenous gene NbNRC3, enabling it to overcome the immunosuppression caused by SS15 secreted by pathogens. These results demonstrate that GRAPE is a highly efficient and universal directed evolution system platform capable of generating superior gene variants, thus contributing to future agricultural breeding.

[0044] Solution for solving the problem

[0045] [1]. A controlled artificial replicon of a geminivirus, comprising two long intergenic regions (LIRs) derived from common soybean yellow dwarf virus (BeYDV), with the two LIRs located on either side of the artificial replicon.

[0046] Optionally, the target gene to be replicated is contained between the two LIRs.

[0047] Optionally, the nucleotide sequence of the LIR is selected from at least one of the following groups (i)-(iv):

[0048] (i) Contains a nucleotide sequence as shown in SEQ ID NO:1;

[0049] (ii) A mutant sequence of the nucleotide sequence shown in SEQ ID NO:1, wherein the mutant sequence has a mutated nucleotide at one or more positions corresponding to the sequence shown in SEQ ID NO:1, and the mutant sequence has the function or activity of the nucleotide sequence shown in SEQ ID NO:1.

[0050] (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii) and has the function or activity of the nucleotide sequence shown in SEQ ID NO:1;

[0051] (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in SEQ ID NO:1.

[0052] [2]. The controlled artificial replicon of the geminivirus according to [1], wherein a short intergenic region (SIR) is included between the two LIRs; or, no SIR is included between the two LIRs.

[0053] [3]. A controlled artificial replicon of a geminivirus according to [1] or [2], wherein the target gene is operatively linked to an expression regulatory sequence.

[0054] [4]. A controlled artificial replicon of a geminivirus according to any one of [1] to [3], wherein the replication of the artificial replicon is controlled by Rep and / or RepA proteins derived from bean yellow dwarf virus (BeYDV);

[0055] Optionally, the replication of the artificial replicon is controlled by the Rep protein derived from common bean yellow dwarf virus (BeYDV);

[0056] Preferably, the replication of the artificial replicon is controlled by the Rep and RepA proteins derived from common bean yellow dwarf virus (BeYDV).

[0057] [5]. According to the controlled artificial replicon of the geminivirus described in [4], wherein the amino acid sequence of the Rep protein is selected from at least one of the group consisting of (i)-(iv):

[0058] (i) Contains an amino acid sequence as shown in SEQ ID NO:3;

[0059] (ii) An amino acid sequence having at least 70%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:3;

[0060] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:3, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:3.

[0061] (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:3 under stringent conditions, and said amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:3, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0062] And / or, the amino acid sequence of the RepA protein is selected from at least one of the following groups (i)-(iv):

[0063] (i) Contains an amino acid sequence as shown in SEQ ID NO:4;

[0064] (ii) An amino acid sequence having at least 70%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:4;

[0065] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:4, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:4.

[0066] (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:4 under stringent conditions, and said amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:4, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0067] [6]. A plant-based directed evolution / screening system for directed evolution or screening of genetic elements to obtain mutants of the genetic elements having a desired function, the plant-based directed evolution / screening system comprising:

[0068] (i) a controlled artificial replicon of a twin virus as described in any of [1] to [5];

[0069] (ii) Rep and / or RepA proteins as described in [4] or [5]; and,

[0070] (iii) Mutants or mutant libraries of the genetic element;

[0071] The replication of the controlled artificial replicon of the twin virus is configured to be associated with the desired function of the mutant of the genetic element.

[0072] [7]. According to the plant-based directed evolution / screening system described in [6], wherein the genetic elements include protein-coding sequences, functional RNA-coding sequences, or expression regulatory sequences; and / or,

[0073] The genetic elements are derived from plants or are intended to be applied to plants.

[0074] [8]. The plant-based directed evolution / screening system according to [6] or [7], wherein the replication of the geminivirus-controlled artificial replicon includes the replication of the geminivirus-controlled artificial replicon in plant cells; and / or,

[0075] The plant-based directed evolution / screening system is used within plant cells;

[0076] Optionally, the plant includes monocotyledonous plants or dicotyledonous plants;

[0077] Optionally, the plants include corn, wheat, rice, barley, sorghum, beans, beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, or tobacco;

[0078] Optionally, the plant cell is an isolated plant cell, a cell in a plant tissue, a cell in a plant organ, or a cell in a plant body.

[0079] [9]. A plant-based directed evolution / screening system according to any one of [6] to [8], wherein the coding sequences of the controlled artificial replicon of the geminivirus, the Rep and / or RepA proteins, and the coding sequences of mutants of genetic elements or mutants in a mutant library are constructed into a vector.

[0080]

[0010] . The plant in vivo directed evolution / screening system according to any one of [6] to [9], wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant in a mutant library;

[0081] Optionally, the mutant library of the genetic element is obtained by inserting multiple mutants of the genetic element into a controlled artificial replicon of a geminivirus.

[0082]

[0011] . The plant-based directed evolution / screening system according to any one of [6] to

[0010] , wherein the replication of the geminivirus controlled artificial replicon is configured to be associated with the desired function of the mutant of the genetic element, comprises: setting the replication level of the geminivirus controlled artificial replicon to be associated with the desired function of the mutant of the genetic element, or setting the activity or expression level of Rep and / or RepA proteins in plant cells to be associated with the desired function of the mutant of the genetic element.

[0083]

[0012] . The plant in vivo directed evolution / screening system according to any one of [6] to

[0011] , wherein the plant in vivo directed evolution / screening system is used for directed evolution of antiviral tRNA, comprising:

[0084] (i) a vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant from a mutant library, wherein the genetic element is an antiviral tRNA to be evolved; and,

[0085] (ii) A vector for expressing Rep and / or RepA proteins, wherein the expression cassette of Rep and / or RepA proteins contains: a tandem start codon (TIC), a stop codon, a self-cleaving peptide coding sequence, and a Rep and / or RepA protein coding sequence.

[0086]

[0013] . The plant in vivo directed evolution / screening system according to any one of [6] to

[0011] , wherein the plant in vivo directed evolution / screening system is used for directed evolution of mutants of immune-related proteins in plants, wherein the mutants of immune-related proteins relieve the inhibition of proteins derived from pathogens, comprising:

[0087] (i) A vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant in a mutant library, wherein the genetic element is the coding sequence of a mutant of the immune-related protein to be evolved;

[0088] (ii) a vector expressing Rep and / or RepA proteins; and,

[0089] (iii) A vector expressing the pathogen-derived protein, which has an inhibitory effect on innate / initiated immune-related proteins;

[0090] Optionally, the immune-related protein includes proteins in the NLR network; preferably, the immune-related protein includes NRC.

[0091] More preferably, the immune-related protein includes NRC3, and the pathogen-derived protein includes SS15.

[0092]

[0014] . According to the plant in vivo directed evolution / screening system described in

[0013] , the coding sequence of the NRC3 mutant to be evolved to relieve SS15 inhibition contains an intron sequence, preferably, the intron sequence contains a DNA barcode.

[0093]

[0015] . The plant in vivo directed evolution / screening system according to any one of [6] to

[0011] , wherein the plant in vivo directed evolution / screening system is selected from any one of the following (A) to (F):

[0094] (A) Plant in vivo directed evolution / screening systems for screening peptides, including:

[0095] (i) A vector expressing a first fusion protein, the first fusion protein comprising the N-terminus of the inteptide to be screened, and the N-terminus or C-terminus of Rep and / or RepA protein;

[0096] (ii) a vector expressing a second fusion protein, the second fusion protein comprising the C-terminus of the inteptide to be screened, and the C-terminus or N-terminus of Rep and / or RepA protein; and,

[0097] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0098] Wherein, the N-terminus or C-terminus of the Rep and / or RepA protein in the vector expressing the first fusion protein and the C-terminus or N-terminus of the Rep and / or RepA protein in the vector expressing the second fusion protein together form a complete Rep and / or RepA protein.

[0099] (B) Plant-based directed evolution / screening systems for screening sequence-specific proteases, including:

[0100] (i) A vector expressing the sequence-specific protease to be screened;

[0101] (ii) a vector expressing a fusion protein, said fusion protein comprising, from N-terminus to C-terminus: a degrader, a peptide recognized by the sequence-specific protease to be screened, and Rep and / or RepA protein; and,

[0102] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0103] (C) Plant-based directed evolution / screening systems for screening sequence-specific recombinases, including:

[0104] (i) A vector expressing Rep and / or RepA proteins, the vector comprising a coding sequence of Rep and / or RepA proteins, and two recombinase recognition sites located at the 5' end of the coding sequence of Rep and / or RepA proteins, with a terminator and a stop codon between the two recombinase recognition sites;

[0105] (ii) A vector expressing the sequence-specific recombinase to be screened, and,

[0106] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0107] (D) Plant-based directed evolution / screening systems for screening self-cleaving peptides, including:

[0108] (i) a vector for expressing a fusion protein, wherein the fusion protein comprises, from the N-terminus to the C-terminus: a degrader, a self-cleaving peptide to be screened, and Rep and / or RepA protein; and,

[0109] (ii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0110] (E) Plant-based directed evolution / screening systems for screening transcription activators, including:

[0111] (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by a known DNA-binding domain, a recognition sequence of the transcription activator to be screened, and a sequence encoding Rep and / or RepA proteins.

[0112] (ii) a vector expressing a fusion protein, said fusion protein comprising the selected transcription activator and the known DNA-binding domain, and,

[0113] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0114] (F) Plant-based directed evolution / screening systems for screening DNA-binding proteins, including:

[0115] (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by the DNA-binding domain to be screened, a recognition sequence of a known transcription activator, and a sequence encoding Rep and / or RepA proteins.

[0116] (ii) a vector for expressing a fusion protein, said fusion protein comprising the known transcription activator and the DNA-binding domain to be screened, and,

[0117] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0118]

[0016] . The plant in vivo directed evolution / screening system according to any one of [6] to

[0011] , wherein the plant in vivo directed evolution / screening system is selected from any one of (G) to (I):

[0119] (G) Plant-based directed evolution systems for screening DNA recombination factors, including:

[0120] (i) Vectors expressing DNA recombinant factors to be evolved;

[0121] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0122] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0123] (H) Plant-based directed evolution systems for screening DNA deaminases, including:

[0124] (i) A vector expressing DNA deaminases to be evolved;

[0125] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0126] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0127] (I) A plant-based directed evolution system for screening plant immune elicitors, including:

[0128] (i) Vectors expressing plant immune elicitors to be evolved;

[0129] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0130] (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

[0131]

[0017] . A method for directed evolution / screening in plants, wherein the method employs a directed evolution / screening system for plants as described in any one of [6] to

[0016] to perform directed evolution or screening of genetic elements to obtain mutants of the genetic elements having the desired function.

[0132]

[0018] . The plant in vivo directed evolution / screening method according to

[0017] includes:

[0133] The steps of introducing the plant intracellular directed evolution / screening system into a population of plant cells; and,

[0134] The steps for culturing the population of plant cells;

[0135] Preferably, the plant-based directed evolution / screening system is introduced into the plant cell population via Agrobacterium infiltration.

[0136]

[0019] . The plant in vivo directed evolution / screening method according to

[0017] or

[0018] further includes:

[0137] The steps include detecting and selecting genetic element mutants with the desired function in a population of said plant cells, and optionally, identifying the function of the selected genetic element mutants.

[0138]

[0020] . A genetic element mutant, obtained by using the plant-based directed evolution / screening system as described in any one of [6] to

[0016] , or by the plant-based directed evolution / screening system method as described in any one of

[0017] to

[0019] .

[0139]

[0021] . The genetic element mutant according to

[0020] is an antiviral tRNA, wherein the nucleotide sequence of the antiviral tRNA is selected from at least one of the following groups (i)-(iv):

[0140] (i) Contains a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153;

[0141] (ii) A mutant sequence of a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, wherein the mutant sequence has a mutated nucleotide at one or more positions of the sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, and the mutant sequence has the function or activity of the nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153;

[0142] (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153.

[0143] (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153, and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153.

[0144]

[0022] . According to the genetic element mutant described in

[0021] , the virus is a virus whose replication and / or transmission are affected by stop codon readout;

[0145] Optionally, the virus includes at least one of the following families: Virgaviridae, Tombusviridae, Benyviridae, Luteoviridae, and Solemoviridae.

[0146] Preferably, the virus includes at least one virus from the family Virgaviridae and the family Tobusviridae;

[0147] More preferably, the virus includes at least one of Tobacco mosaic virus (TMV), Turnip vein-clearing virus (TVCV), Tobacco rattle virus (TRV), and Tomato bushy stunt virus (TBSV).

[0148]

[0023] . A biomaterial selected from any one of the following (a) to (d):

[0149] (a) An isolated polynucleotide, wherein the polynucleotide encodes the antiviral tRNA described in

[0021] or

[0022] ;

[0150] (b) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide described in (a);

[0151] (c) A vector, wherein the vector comprises the polynucleotide described in (a) or the nucleic acid construct described in (b);

[0152] (d) Recombinant host cell, wherein the recombinant host cell comprises the antiviral tRNA described in

[0021] or

[0022] , the polynucleotide described in (a), the nucleic acid construct described in (b), or the vector described in (c); preferably, the host cell is derived from a plant.

[0153]

[0024] . The following uses of the antiviral tRNA as described in

[0021] or

[0022] , or the biological material as described in

[0023] : for antiviral use in plants; and / or, for use in the preparation of reagents for antiviral use in plants.

[0154]

[0025] . A method for improving plant resistance to viruses, the method comprising the steps of introducing antiviral tRNA as described in

[0021] or

[0022] , and / or biological material as described in

[0023] into the plant.

[0155]

[0026] . The genetic element mutant according to

[0020] is a mutant of NRC3, which, compared with wild-type NRC3, reduces or relieves the immunosuppression of SS15 secreted by pathogens in plants;

[0156] Optionally, the mutant is selected from any one of the following groups (i)-(ii);

[0157] (i) A mutant comprising the sequence shown in SEQ ID NO:10, wherein the mutant has a mutated amino acid at one or more positions from position 308 to 383 of the sequence shown in SEQ ID NO:10;

[0158] (ii) A polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (i);

[0159] Preferably, the mutant has a mutated amino acid at one or more positions in the sequence shown in SEQ ID NO:10, from position 308 to 346;

[0160] More preferably, the mutant corresponds to the sequence shown in SEQ ID NO:10 and has a mutated amino acid at any one position, or at any two or more positions, of the group consisting of (a1)-(a5):

[0161] (a1) The 312th amino acid, preferably K312P;

[0162] (a2) The 315th amino acid is preferably T315W;

[0163] (a3) The 316th amino acid is preferably E316R, E316K, or E316P;

[0164] (a4) The 317th amino acid is preferably N317K;

[0165] (a5) The 320th amino acid, preferably W320F.

[0166]

[0027] . The genetic element mutant according to

[0026] , wherein the mutant corresponds to the sequence shown in SEQ ID NO:10, and further has a mutated amino acid at the following position (a6):

[0167] (a6) Amino acid at position 480, preferably D480V.

[0168]

[0028] . The genetic element mutant according to

[0027] , wherein the mutant corresponds to the sequence shown in SEQ ID NO:10 and has the following (m1)-(m 12 Any of the mutated amino acids shown in )

[0169] (m1)E316R;

[0170] (m2)E316K;

[0171] (m3)T315W;

[0172] (m4)K312P;

[0173] (m5)E316P+W320F;

[0174] (m6)N317K+W320F;

[0175] (m7)E316R+D480V;

[0176] (m8)E316K+D480V;

[0177] (m9)T315W+D480V;

[0178] (m 10 K312P+D480V;

[0179] (m 11 E316P+W320F+D480V;

[0180] (m 12 )N317K+W320F+D480V.

[0181]

[0029] . A biomaterial selected from any one of the following (a) to (d):

[0182] (a) An isolated polynucleotide, wherein the polynucleotide encodes a mutant of NRC3 as described in any one of [26-28];

[0183] (b) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide described in (a);

[0184] (c) A vector, wherein the vector comprises the polynucleotide described in (a) or the nucleic acid construct described in (b);

[0185] (d) Recombinant host cell, wherein the recombinant host cell comprises a mutant of NRC3 as described in any one of

[0026] to

[0028] , a polynucleotide as described in (a), a nucleic acid construct as described in (b), or a vector as described in (c); preferably, the host cell is derived from a plant.

[0186]

[0030] . The use of a mutant of NRC3 as described in any one of

[0026] to

[0028] , or the use of the biological material as described in

[0029] in at least one of the following (a)-(d):

[0187] (a) Use in reducing or relieving immunosuppression of SS15 secreted by pathogens in plants;

[0188] (b) Use in the preparation of reagents for reducing or relieving immunosuppression of SS15 secreted by pathogens in plants;

[0189] (c) Uses in enhancing plant resistance to pathogens;

[0190] (d) Use in the preparation of reagents for enhancing plant resistance to pathogens;

[0191] Preferably, the pathogen includes secreted SS15 pathogens, such as potato white nematode.

[0192]

[0031] . A method for improving plant resistance to pathogens, the method comprising the steps of introducing a mutant of NRC3 as described in any one of

[0026] to

[0028] , and / or biological material as described in

[0029] into the plant.

[0193]

[0032] . A kit comprising a controlled artificial replicon of a geminivirus as described in any one of [1] to [5], or a plant-based directed evolution / screening system as described in any one of [6] to

[0016] .

[0194]

[0033] . The application of genetic element mutants obtained by using the plant-based directed evolution / screening system as described in any one of [6] to

[0016] , or by the plant-based directed evolution / screening system method as described in any one of

[0017] to

[0019] , in plants, especially in plant genetic engineering.

[0195] The effects of the invention

[0196] I. In some embodiments of this invention, geminiviruses are modified into plant artificial replicons. This invention collected 19 geminiviruses belonging to three genera and modified them into self-replicating and controlled artificial replicons. Among the many viruses, Common Bean Yellow Dwarf Virus (BeYDV) has the strongest replication ability; therefore, subsequent experiments were based on the replicons of this virus. This invention also constructed a reporter replicon, raGFP, to indicate the level of rolling circle replication.

[0197] II. In some embodiments of this invention, GRAPE, a universal, rapid, and high-throughput directed evolution system within plants, was established. This invention demonstrates that GRAPE can withstand 10 [units of measurement unclear] in a single leaf of *Nicotiana benthamiana*. 5The invention demonstrates that GRAPE can achieve a stable library size and, under these conditions, maintain a stable proportion of each library member. It also proves that GRAPE can screen target genes at this throughput within 4 days. Finally, the invention uses a series of examples to demonstrate that the expected functions of various target genes can be positively or negatively coupled with geminivirus rolling circle replication, indicating that these genes can all be targets for GRAPE evolution. These results demonstrate that GRAPE is a universal, rapid, and high-throughput directed evolution system for plants, far faster and more efficient than other current directed evolution systems for plants.

[0198] III. In some embodiments of this invention, a series of anti-viral tRNAs were obtained through GRAPE phylogenetic analysis, and it was demonstrated that they can inhibit viral replication in plants. Targeting the gene expression strategies of the Cladoviridae and Tomato Cladoviridae families, this invention hypothesizes that tRNAs promoting stop codon reading can inhibit the replication of these two types of viruses. By coupling the function of anti-viral tRNAs with rolling circle replication of geminivirus replicons, this invention evolved anti-viral tRNAs capable of achieving efficient stop codon reading. Subsequently, this invention demonstrated that anti-viral tRNAs can inhibit the replication of TMV, TVCV, TBSV, and TRV in plants, and inhibit the systemic spread of TMV in plants. Subsequent experiments demonstrated that anti-viral tRNAs achieve antiviral activity by promoting stop codon reading in the viral genome.

[0199] IV. In some embodiments of the present invention, NRC3 variants unrepressed by SS15 were obtained using GRAPE evolution. SS15 can inhibit the formation of NRC3 pentamers by directly binding to NRC3, thereby hindering its function. Through domain interchange experiments, the present invention suggests that SS15 may interact with the HD1-1 domain of NRC3. Next, the present invention performed saturation mutagenesis on this region and negatively coupled NRC3 function to replicon rolling loop replication. Through GRAPE directed evolution, the present invention obtained a series of unrepressed NRC3 variants. These variants can respond to a range of plant disease resistance signals. The present invention also demonstrates that these variants are unrepressed by SS15 by avoiding interaction with it.

[0200] The results above demonstrate that GRAPE is a universal, rapid, and efficient system for directed evolution within plants, which can be used to generate new superior alleles and assist in future agricultural breeding. Attached Figure Description

[0201] Figure 1: The process of releasing the Gemini virus replicon from T-DNA.

[0202] To release the geminivirus replicon from Agrobacterium T-DNA, this invention inserts two LIR copies onto the flanks of the replicon sequence. Upon transformation into plant cells, the T-DNA first transforms into double-stranded DNA. Subsequently, the cis- or trans-expressed Rep protein cleaves the neck-loop structure in the LIR and recruits endogenous DNA polymerase to initiate DNA replication. The single-stranded DNA released during replication can then be cleaved and ligated by the Rep protein, thereby circularizing into a circular DNA replicon.

[0203] Figure 2: Development of a self-replicating artificial replicon for twin viruses.

[0204] Figure 2A shows a schematic diagram of the construction of self-replicating artificial replicons. The replication proteins of the geminiviruses (Rep / RepA for *Maize Stripe Virus*, and C1 to C4 for *Bean Golden Mosaic Virus* and *Beetroot Curly Top Virus*) are retained in the replicons, while the viral strand genes (mobility protein MP and capsid protein CP) are replaced by a GFP expression frame. Figure 2B shows the copy number levels of self-replicating artificial replicons from different geminiviruses. In this invention, the corresponding vectors were transformed into *Tobacco Bengal* leaves using the Agrobacterium infiltration method, and the replicon copy number was determined by qPCR after 4 days. In Figure 2B, values ​​are mean ± sem and based on 6 independent biological replicates. Statistical methods used were multiple comparisons Tukey's test based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0205] Figure 3: The working status of BeYDV controlled replicon construction.

[0206] Figure 3A shows a schematic diagram of the construction of controlled artificial replicons. Based on the BeYDV self-replicating artificial replicons, this invention constructed nine controlled replicons. To control rolling circle replication, the replication proteins Rep and RepA were removed from the replicons and expressed in trans form. Figure 3B shows the copy number levels of the nine controlled replicons. The corresponding vectors were transformed into *Nicotiana benthamiana* leaves using the Agrobacterium infiltration method, and the replicon copy number was determined by qPCR after 4 days. In Figure 3B, values ​​are mean ± sem and are based on 6 independent biological replicates. Statistical analysis was performed using a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0207] Figure 4: Development of controlled artificial replicons of geminiviruses. Figure 4A shows a schematic diagram of the construction of controlled artificial replicons. Open reading frames in all viral genomes were removed and replaced with a GFP expression frame. Viral replication proteins were expressed by a separate vector. Figure 4B shows the copy number levels of controlled artificial replicons from different geminiviruses. In this invention, the corresponding vectors were transformed into tobacco leaves using Agrobacterium tumefaciens infiltration. Viral replication proteins were injected with or without the vectors. Replicon copy numbers were determined by qPCR after 4 days. In Figure 4B, values ​​are mean ± sem and are based on 6 independent biological replicates. Statistical methods used were Tukey's test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0208] Figure 5: Schematic diagram and working status of the report replicon raGFP.

[0209] Figure 5A shows a schematic diagram of the construction and working principle of the reporter replicon raGFP. To avoid leakage expression of the reporter gene GFP when rolling circle replication does not occur, this invention divides GFP into N-terminal and C-terminal parts. Only when rolling circle replication occurs, a circular replicon is formed, and GFP can then be expressed normally through transcriptional splicing. Figure 5B shows the working state of the reporter replicon raGFP. No green fluorescence was observed without the addition of Rep / RepA; strong green fluorescence was observed with the addition of Rep / RepA. Leaf samples of *Nicotiana benthamiana* were photographed under UV light three days after *Agrobacterium* injection. This experiment was performed in three independent biological replicates and similar results were obtained.

[0210] Figure 6: Copy number levels and stability of replicons of different sizes.

[0211] Figure 6A shows the copy number levels of replicons of different sizes. Replicons of different sizes were co-injected into *Nicotiana benthamiana* leaves with or without Rep / RepA. Copy numbers were determined by qPCR 4 days post-injection. Figure 6B shows the replication stability of replicons of different sizes. In this invention, sample DNA was subjected to high-throughput sequencing, and the sequencing reads were then coupled to the replicons to calculate relative coverage. Values ​​in the figures are mean ± sem (Figure 6A) or mean (Figure 6B), both based on three independent biological replicates. Statistical methods used were Tukey's test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figures represent statistically distinct groups (P < 0.05).

[0212] Figure 7: Schematic diagram of non-screening replicon libraries and flowchart for determining experimental throughput.

[0213] To determine the experimental throughput of tobacco leaves, this invention inserts a diverse barcode DE containing a series of discontinuous degenerate bases into a replicon using Golden Gate assembly, constructing a non-selective replicon library. By adjusting the number of degenerate bases used for analysis, the library size can be adjusted. This non-selective replicon library is then sequentially transformed into *E. coli* and *Agrobacterium*, and analyzed at different OD values. 600 Values ​​were converted and injected into tobacco leaves (one leaf per experiment). Constitutively expressed Rep / RepA was also injected. Four days later, the tobacco leaves were collected and deep sequenced. The centrifuge tubes in the figure represent the stages of sample collection and deep sequencing. For the throughput that a tobacco leaf can handle, the proportion of all barcodes in the library should remain constant, and vice versa.

[0214] Figure 8: Non-screened libraries in different OD 600 Performance under different values ​​and library sizes.

[0215] Figures 8A and 8B show examples of the proportion of each variant in a non-selective replicon library before and after Agrobacterium injection. The x-axis and y-axis represent the proportion of each variant in the initial library ligation product and the final replicon, respectively. The color of a dot in the figure represents the number of surrounding dots. Figure 8C shows the OD values ​​of non-selective replicon libraries of different library sizes at different Agrobacterium injection sites. 600 The working condition under the given value. R in the diagram. 2 The value is calculated based on the proportion of each variant in the initial library ligation product and the final replicon. The number shown at the center of the point in the figure is the exact R value. 2 Value. The red dashed line in the diagram is based on R. 2 The value was calculated to be equal to 0.95. The values ​​in C in Figure 8 are all averages and are based on three independent biological replicates.

[0216] Figure 9: Schematic diagram of screening replicon library and working principle of screening in plant.

[0217] To test the feasibility of in vivo screening in plants, this invention inserts Rep / RepA into a geminivirus replicon and mutates its DNA-binding motif I into three variants: a functional variant, a non-functional variant, and an unclassified variant. This invention also inserts a diversification barcode into the replicon to regulate the library size. This invention then uses this screening replicon library with different OD values. 600One leaf was injected into leaves of *Nicotiana benthamiana* per experiment, and the leaves were collected 4 days later for deep sequencing. Replicons containing functional Rep / RepA variants can undergo rolling circle replication, thus increasing their proportion in the replicon library. Conversely, the opposite is also true.

[0218] Figure 10: Screening libraries in different OD 600 Performance under different values ​​and library sizes.

[0219] Figures A and B in Figure 10 show examples of the proportion of each variant in the selected replicon library before and after Agrobacterium injection. The x-axis and y-axis represent the proportion of each variant in the initial library ligation product and the final replicon, respectively. The color of the points in the figure is determined according to the variant type. When Agrobacterium is injected with OD... 600 When the value is low (A in Figure 10), the screening strength is strong; while when Agrobacterium is injected with OD 600 At higher values ​​(B in Figure 10), the screening strength weakens. This is because the high concentration of Agrobacterium causes a hitch effect. Figure 10, C, shows the screening replicon libraries of different library sizes at different Agrobacterium injection OD values. 600 The working condition under the given value. R in the diagram. 2 The value is calculated based on the proportion of each variant in the initial library ligation product and the final replicon. The number shown at the center of the point in the figure is the exact R value. 2 Value. The red and blue dashed lines in the graph are based on R respectively. 2 Values ​​less than 0.12 and coverage greater than 0.5 are plotted. The values ​​in C in Figure 10 are all averages and are based on three independent biological replicates.

[0220] Figure 11: Agrobacterium OD 600 The relationship between value and conversion efficiency and conversion multiplicity.

[0221] Figure 11A shows a schematic diagram of a dual-fluorescence experiment used to evaluate the multiple transformation efficiency of Agrobacterium. To eliminate the different OD values ​​of Agrobacterium... 600 To differentiate gene expression levels, green fluorescent protein (GFP) and red fluorescent protein (mScarlet) were constructed into self-replicating replicons of BeYDV. Replicons containing GFP and mScarlet were then transformed into Agrobacterium EHA105, respectively. The two strains were mixed in equal proportions and injected into Nicotiana benthamiana at a concentration gradient. Figure 11, B, shows the results of the dual-fluorescence assay. At OD... 600 When the value is between 0.001 and 0.003, the red and green fluorescence are completely separated; when the OD... 600 When the value is between 0.01 and 0.03, the fluorescence overlaps; while when the OD value is between 0.01 and 0.03, the fluorescence overlaps. 600When the value is higher than 0.1, the fluorescence completely overlaps. Three days after injection, tobacco mesophyll cells were photographed under a stereofluorescence microscope. This experiment was performed three independent biological replicates, yielding similar results. The scale bar is 500 μm. Figure 11C shows a schematic diagram of the gene-editing experiment used to evaluate Agrobacterium-mediated transformation efficiency. Since gene-editing efficiency is directly related to Agrobacterium-mediated transformation efficiency, this invention designed a gene-editing experiment to evaluate different OD values. 600 The transformation efficiency of Agrobacterium was measured. This invention uses SpCas9 and sgRNA targeting the tobacco SGS3 gene for gene editing of the tobacco genome. To eliminate different Agrobacterium OD values... 600 Based on the difference in gene expression levels, sgRNA was constructed into the self-replicating replicon of BeYDV. Agrobacterium tumefaciens used sgRNA in a concentration gradient with 0.2 OD... 600 35Sp-SpCas9 was co-injected into *Nicotiana benthamiana*. Figure 11 shows the results of the gene editing experiment (D). When OD... 600 At values ​​of 1.0 and 0.3, gene editing efficiency is at a very high level. When OD... 600 When the value is between 0.01 and 0.1, the gene editing efficiency is at an acceptable level. And when the OD value is... 600 When the value is less than 0.003, the gene editing efficiency becomes very low. In D of Figure 11, all values ​​are mean ± sem and are based on 6 independent biological replicates. The statistical method used was a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0222] Figure 12: Forward coupling of the function of the target gene with the rolling circle replication of the replicon.

[0223] Figure 12A shows a schematic diagram of the forward coupling of the function of the target gene with the rolling circle replication of the replicon. This invention designs a genetic pathway based on the expected biological function of the target gene and couples it with the rolling circle replication of a geminivirus artificial replicon. Thus, a functional target gene variant can initiate the expression of Rep / RepA, thereby triggering the rolling circle replication of the replicon. Figure 12B shows the forward coupling of the integument with the rolling circle replication of the replicon. To forward couple the integument with rolling circle replication, this invention splits Rep / RepA into two parts at C183 and fuses its N-terminus and C-terminus to N-integuments and C-integuments, respectively. The functional and mutually recognizing N-integuments and C-integuments can link the two parts of Rep / RepA together, thereby initiating the rolling circle replication of the replicon. This invention injects the Rep / RepA fragment fused with N-integuments and C-integuments, along with a raGFP reporter replicon, into tobacco leaves. The replicon copy number is measured 4 days after injection. The numerical averages in B of Figure 12 are based on four independent biological replicates. C of Figure 12 shows the forward coupling of the sequence-specific protease to the rolling circle replication of the replicon. In this invention, a degrader is fused to the N-terminus of Rep / RepA via a cleavage peptide of a sequence-specific protease. The degrader mediates the degradation of Rep / RepA in plant cells, and the protease capable of cleaving this peptide releases the degrader, thereby stabilizing Rep / RepA and subsequently initiating rolling circle replication of the replicon. In this invention, a sequence-specific protease is co-injected with Rep / RepA containing the degrader and protease recognition sequence fused to the N-terminus, along with a raGFP reporter replicon, into tobacco leaves. The replicon copy number is measured four days after injection. The numerical averages in C of Figure 12 are based on four independent biological replicates. D of Figure 12 shows the forward coupling of the recombinase to the rolling circle replication of the replicon. In this invention, two recombinase recognition sites are inserted at the front of the Rep / RepA coding sequence, and a terminator is inserted between the two sites. This terminator inhibits Rep / RepA expression, while a recombinase capable of recognizing the recombination site can delete this terminator, activating Rep / RepA expression and subsequently initiating replicon rolling circle replication. In this invention, the recombinase and Rep / RepA containing the recombination site, along with a raGFP reporter replicon, are co-injected into *Nicotiana benthamiana* leaves. Replicon copy number is measured 4 days after injection. The numerical values ​​in D of Figure 12 are averages, based on 4 independent biological replicates. E of Figure 12 shows the forward coupling of the self-cleaving peptide to replicon rolling circle replication. In this invention, a degradogen is fused to the N-terminus of Rep / RepA via a self-cleaving peptide. The degradogen mediates the degradation of Rep / RepA in plant cells, and the self-cleaving peptide releases the degradogen, thereby stabilizing Rep / RepA and subsequently initiating replicon rolling circle replication.This invention involves injecting Rep / RepA, fused with a degrader and a self-splicing peptide, along with a raGFP reporter replicon into *Nicotiana benthamiana* leaves. Replicon copy number was measured 4 days after injection. Values ​​in Figure 12 (E) are mean ± sem and based on three independent biological replicates. Statistical analysis was performed using a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05). Figure 12 (F) shows a schematic diagram of the positive coupling of transcription activator and rolling circle replication of the replicon. This invention fuses a transcription activator with the DNA-binding domain of GAL4 and drives Rep / RepA expression using a basal promoter with six UAS tandem repeats upstream. Only functional transcription activators can initiate Rep / RepA expression, thereby driving rolling circle replication of the reporter replicon. This invention involves injecting a GAL4-fused transcription activator, Rep / RepA, and raGFP reporter replicon together into *Nicotiana benthamiana* leaves. Replicon copy number was measured 4 days after injection. The values ​​in F in Figure 12 are mean ± sem and are based on three independent biological replicates. Statistical analysis was performed using a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05). G in Figure 12 is a schematic diagram illustrating the forward coupling of the DNA-binding protein with the rolling circle replication of the reporter replicon. This invention fuses the DNA-binding protein with the ERF2-AD activator and drives Rep / RepA expression using a basal promoter with an upstream tandem repeat of the DNA-binding sequence. Rep / RepA expression is only initiated when the DNA-binding protein binds to the promoter region, thereby driving the rolling circle replication of the reporter replicon. In this invention, the DNA-binding protein fused with the activation domain, along with Rep / RepA and the raGFP reporter replicon, is injected co-injected into *Nicotiana benthamiana* leaves. Replicon copy number was measured four days after injection. The values ​​in G in Figure 12 are mean and are based on four independent biological replicates.

[0224] Figure 13: Negative coupling of the function of the target gene with the rolling circle replication of the replicon.

[0225] Figure 13A illustrates the negative coupling of the intended function of the target gene with replicon rolling circle replication. Specific types of target genes can directly or indirectly interfere with the process of geminivirus rolling circle replication; therefore, when constitutively expressed Rep / RepA is present, its functional variant can inhibit replicon rolling circle replication. Figure 13B shows the negative coupling of the function of homologous recombination factors with replicon rolling circle replication. Figure 13C shows the negative coupling of the function of DNA deaminase with replicon rolling circle replication. Figure 13D shows the negative coupling of the function of plant immune elicitors with replicon rolling circle replication. The corresponding vectors were transformed into *Nicotiana benthamiana* leaves using the Agrobacterium infiltration method. Leaf samples were collected 4 days post-transformation, and replicon copy number was determined. In Figures 13B, C, and D, values ​​are mean ± sem and are based on 4 (Figure 13B and C) or 6 (Figure 13D) independent biological replicates. Statistical methods used were multiple comparisons Tukey's test based on one-way ANOVA. The lowercase letters in the figure represent statistically distinct groups (P<0.05).

[0226] Figure 14: GRAPE workflow.

[0227] In GRAPE, this invention first mutagenesis of the target gene GOI in vitro, and then inserts its product into a geminivirus replicon to construct a GRAPE library. Next, this invention designs a genetic pathway to couple the expected function of the target gene product with geminivirus rolling circle replication (RCR) to achieve screening of the target gene in plants. Subsequently, this invention injects the library into leaves of *Nicotiana benthamiana*, and extracts leaf DNA four days later for deep sequencing of the target gene. Thus, by calculating the change in the proportion of each target gene variant in the GRAPE library before and after *Agrobacterium* injection, and through iteration, this invention can obtain target gene variants with the expected function.

[0228] Figure 15: Schematic diagram of the structure of four viruses carrying GFP.

[0229] The structural diagrams of infectious clones of TMV, TVCV, TBSV, and TRV carrying GFP are shown. The red arrows in the diagrams indicate the leaky stop codons in the viral RdRp.

[0230] Figure 16: Verification of the effect of the SRS / RdRp ratio on viral replication.

[0231] Figure 16A shows a schematic diagram of the viral stop codon substitution experiment. To investigate the effect of the SRS / RdRp ratio on viral replication, the leaky stop codons in the RdRp of TVCV and TBSV viruses were replaced with TAA or TGG. The TAA stop codon reduced readthrough efficiency, while the TGG tryptophan codon improved it. Figure 16B shows that normal readthrough of RdRp is crucial for viral replication. The corresponding vectors were transformed into leaves of *Nicotiana benthamiana* using the Agrobacterium-mediated transformation method. Leaves were photographed under UV light 3 days (TBSV) or 4 days (TVCV) after injection. The experiment was performed in three independent biological replicates and yielded similar results.

[0232] Figure 17: The principle of anti-viral tRNA inhibiting viral replication.

[0233] Viruses of the families Cladoviridae and Tomato Cladoviridae rely on a leaky stop codon in their RdRp to express two genes. The ratio of SRS to RdRp is crucial for viral replication. Anti-viral tRNA can promote the reading through this stop codon, interfering with viral replication and thus achieving resistance to the virus.

[0234] Figure 18: Reading efficiency of plant endogenous and mammalian repressive tRNAs.

[0235] Figure 18A shows a schematic diagram of the construction of Termination-FLuc. A stop codon and a P2A self-cleaving peptide are inserted into the N-terminus of firefly luciferase (FLuc). A tandem start codon (TIC) is also introduced to avoid potential ribosome leakage scanning. Only tRNAs that can induce stop codon readthrough can initiate FLuc expression. Figure 18B shows a schematic diagram of a dual-luciferase-based readthrough reporter system. Termination-FLuc detects the readthrough efficiency of tRNAs, while constitutively expressed RLuc serves as an internal control, representing the overall gene expression level in tobacco. TGG-FLuc serves as a positive control. The ratio of the sample FLuc / RLuc value to the positive control is used as the readthrough efficiency of the tRNA. Figure 18C shows the readthrough efficiency of plant endogenous and mammalian repressive tRNAs. In this invention, tRNA and Termination-FLuc are co-injected into leaves of *Nicotiana benthamiana*. Constitutively expressed RLuc is also injected as an internal control. The FLuc / RLuc value is measured 4 days after injection. In Figure 18, B, the values ​​are mean ± sem and are based on 3 independent biological replicates.

[0236] Figure 19: Stop codon readout-dependent sequence environment of plant endogenous repressive tRNAs.

[0237] Figure 19A shows a schematic diagram of the vector used to detect environment-dependent readthrough of the endogenous sup-tRNA sequence. In this invention, a 48bp segment surrounding the stop codon in the viral RdRp is inserted before the coding sequence of FLuc to simulate the viral sequence environment. Figure 19B shows the co-transformation of these vectors with constitutively expressed RLuc into *Nicotiana benthamiana* leaves. The FLuc / RLuc ratio was measured 4 days post-injection. In Figure 19B, all values ​​are mean ± sem and are based on three independent biological replicates. Statistical analysis was performed using a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0238] Figure 20: Construction and operation of Termination-Rep. Figure 20A shows the construction of Termination-Rep. A stop codon and P2A self-cleaving peptide were inserted into the N-terminus of Rep / RepA. A tandem start codon (TIC) was also introduced to avoid potential ribosome leakage scanning. Only tRNAs capable of triggering stop codon readthrough can initiate Rep / RepA expression, thereby initiating rolling circle replication. Figure 20B shows the operation of Termination-Rep. In this invention, termination-FLuc and termination-Rep were co-injected with different tRNAs into *Nicotiana benthamiana*. Replicon copy number and FLuc / RLuc values ​​were measured 4 days post-injection. In Figure 20B, values ​​are mean ± sem and based on three independent biological replicates. Statistical methods used were multiple comparisons Tukey's test based on one-way ANOVA. Lowercase letters in the figure represent statistically distinct groups (P < 0.05).

[0239] Figure 21: The principle of directed evolution of anti-viral tRNA. In this invention, tRNA is mutagenized and inserted into an artificial replicon of a geminivirus to construct a GRAPE library. This library, along with termination-Rep containing different stop codons, is then co-injected into *Nicotiana benthamiana*. tRNAs capable of inducing stop codon readthrough can initiate the expression of Rep / RepA, subsequently initiating their own rolling circle replication, thereby enriching themselves in the replicon pool.

[0240] Figure 22: Directed evolution of anti-viral tRNA and its results.

[0241] Figure 22A shows a schematic diagram of the tRNA library used for the first round of anti-viral tRNA evolution. This invention mutagenesis of 30 high-copy tRNAs found in Solanaceae plants and conversion of their anticodons to CCA, CUA, UCA, and UUA. A total of 1728 tRNAs were constructed into geminivirus replicons, forming the first anti-viral tRNA library. Figure 22B shows the results of the first round of anti-viral tRNA evolution. This invention co-injected the first anti-viral tRNA library into leaves of *Nicotiana benthamiana* using three different termination-Rep vectors, and extracted DNA four days later for deep sequencing. Figure 22C shows a schematic diagram of the tRNA library used for the second round of anti-viral tRNA evolution. This invention further mutagenesis of the five tRNA backbones that performed well in the first round of evolution, and constructed a total of 8192 tRNAs into geminivirus replicons, forming the second anti-viral tRNA library. Figure 22D shows the results of the second round of anti-viral tRNA evolution. In this invention, a second anti-viral tRNA library was co-injected into tobacco leaves using three different termination-Rep vectors. In Figures 22, B and D, the x-axis and y-axis represent the proportion of each tRNA variant in the initial library and replicon, respectively. The color of each point in the figure is determined according to its anticodon type. All data in the figure are averages and are based on three independent biological replicates.

[0242] Figure 23: Readthrough efficiency of Anti-viral tRNA.

[0243] Figure 23A shows the readthrough efficiency of the anti-viral tRNA for the UAG stop codon. Figure 23B shows the readthrough efficiency of the anti-viral tRNA for the UGA stop codon. Figure 23C shows the readthrough efficiency of the anti-viral tRNA for the UAA stop codon. In this invention, the anti-viral tRNA was co-injected into tobacco leaves with three termination-Fluc vectors and constitutively expressed RLuc. The FLuc / RLuc ratio was measured 4 days after injection. All values ​​in the figures are mean ± sem and are based on three independent biological replicates. The statistical method was a Tukey test for multiple comparisons based on one-way ANOVA. Lowercase letters in the figures represent statistically distinct groups (P < 0.05).

[0244] Figure 24: Predicted secondary structures of some evolutionarily derived anti-viral tRNAs.

[0245] The figure shows the predicted secondary structures of four anti-viral tRNAs evolved via GRAPE. Three of them can read the UAG stop codon, and one can read the UGA stop codon. Blue bases represent anticodons, while green bases represent bases different from the original tRNA. The secondary structures of the tRNAs were predicted using RNAstructure software.

[0246] Figure 25: Anti-viral tRNA can promote the readthrough of the stop codon in RdRp.

[0247] Figure 25A shows a schematic diagram of a dual-luciferase experiment simulating viral stop codon readout. In this invention, a 48bp segment near the stop codon in the RdRp of TMV, TVCV, TBSV, and TRV viruses was constructed between RLuc and FLuc. The ratio of luciferase activity in FLuc and RLuc can, to some extent, represent the ratio between viral RdRp and SRS. Figure 25B shows that anti-viral tRNA can promote the readout of the stop codon in viral RdRp. In this invention, anti-viral tRNA and a dual-luciferase reporter vector were co-injected into leaves of *Nicotiana benthamiana*. Luciferase activity was measured after 4 days. In Figure 25B, all values ​​are mean ± sem and are based on three independent biological replicates. P-values ​​were obtained from a one-way ANOVA multiple comparison Tukey test.

[0248] Figure 26: Anti-viral tRNA significantly reduced the expression of virus-encoded GFP.

[0249] Antiviral tRNA can significantly reduce the expression of plant virus-encoding genes. In this invention, infectious clones of TMV, TVCV, TRV, and TBSV carrying GFP, along with natural tRNA or antiviral tRNA, were transformed into *Nicotiana benthamiana* leaves using the Agrobacterium infiltration method. GFP fluorescence in the leaves was captured 2 days (TRV), 3 days (TBSV), or 4 days post-injection (TMV and TVCV). This experiment was performed in three independent biological replicates, yielding similar results.

[0250] Figure 27: Anti-viral tRNA significantly inhibits viral replication.

[0251] Antiviral tRNAs inhibited viral accumulation in plant cells. Infectious clones of TMV, TVCV, TRV, and TBSV carrying GFP were transformed into *Nicotiana benthamiana* leaves via Agrobacterium infiltration along with native tRNA or antiviral tRNA. Viral titers were detected at 2 days (TRV), 3 days (TBSV), or 4 days post-injection (TMV and TVCV). This invention amplified multiple viral genes to assess the titers of individual viral subgenomes. Data in the figures are mean ± sem and are based on three independent biological replicates. P-values ​​were obtained from a one-way ANOVA multiple comparison Tukey test.

[0252] Figure 28: Anti-viral tRNA significantly inhibits systemic viral infection.

[0253] Figure 28A shows a schematic diagram of the TMV-GFP structure. In this invention, the coding sequence of GFP is inserted between the MP and CP of TMV. Figure 28B shows the inhibitory effect of different tRNAs on TMV-GFP systemic infection. In this invention, natural tRNA or anti-viral tRNA is injected into leaves of *Nicotiana benthamiana*, and TMV-GFP is injected at the same location one day later. When green fluorescence is observed on the top leaves of a plant, it is considered to be systematically infected. Each treatment group contains 7 tobacco plants. Figure 28C shows a photograph of a representative tobacco plant. The photograph was taken under ultraviolet light 7 days after TMV-GFP injection.

[0254] Figure 29: Anti-viral tRNA inhibits viral replication by promoting RdRp readthrough.

[0255] Figure 29, A, shows that plant virus replication is not inhibited by mismatched anti-viral tRNAs. The corresponding vectors were injected into *Nicotiana benthamiana* leaves. GFP fluorescence from TRV (whose RdRp contains a UGA stop codon) was not significantly inhibited by anti-viral tRNAs containing a CUA anticodon. Similarly, GFP fluorescence from TMV, TVCV, and TBSV (whose RdRp contains a UAG stop codon) was not significantly inhibited by anti-viral tRNAs containing a UCA anticodon. The results of this orthogonal experiment demonstrate that anti-viral tRNAs inhibit viral replication by promoting the readthrough of the stop codon in the viral RdRp. Figure 29, B, shows that anti-viral tRNAs do not inhibit geminivirus replication or plant gene expression. The corresponding vectors were injected into *Nicotiana benthamiana* leaves. Fluorescence from 35Sp-GFP and BSCTV-GFP was not significantly inhibited by anti-viral tRNAs. These results indicate that anti-viral tRNA does not inhibit viral replication by suppressing plant cell activity. However, this invention cannot rule out the possibility that anti-viral tRNA inhibits viral replication through other pathways. The experiment was performed three independent biological replicates and yielded similar results.

[0256] Figure 30: Inhibition analysis of NRC functional activation by SS15.

[0257] Figure 30A shows that SS15 inhibits NRC3-induced HR. Figure 30B shows the transient expression results of the variant generated by the MHD motif mutation in tobacco. The variant generated by the MHD motif mutation was co-expressed with SS15 in tobacco, and the control group was co-expressed with the empty vector EV. The autonomously replicating BeYDV-GFP replicon was co-expressed in tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated in triplicate, bar = 1 cm.

[0258] Figure 31: Inhibition analysis of SS15 on NRC3-responsive s-NLR immune signaling.

[0259] Results of transient co-expression of NRC3 with Pto / AVRPto or Gpa2 / RBP1 in Nicotiana benthamiana. The treatment group and control group co-expressed SS15 and empty vector (EV), respectively. The autonomously replicating BeYDV-GFP replicon was co-expressed in the tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated three times in parallel, bar = 1 cm.

[0260] Figure 32: Domain exchange experiment of NRC3 and NRC4.

[0261] Figure 32A shows a schematic diagram of the NRC3-NRC4 chimeric protein. Figure 32B shows the transient expression results of the NRC3-NRC4 chimeric protein in tobacco. NRC3D480V, NRC4D478V, and eight other NRC3-NRC4 chimeric proteins were transiently expressed in tobacco. The treatment group and the control group co-expressed SS15 and the empty vector (EV), respectively. The autonomously replicating BeYDV-GFP replicon was co-expressed in tobacco to characterize cell death. Fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated in triplicate, with a bar of 1 cm.

[0262] Figure 33: The effect of NRC3 function activation on the number of replicon copies.

[0263] Figure 33A shows the results of fluorescence observation in tobacco. The autonomously replicating BeYDV-GFP replicon was co-expressed in tobacco to characterize cell death. Fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated in triplicate, with a bar of 1 cm. Figure 33B shows the statistical results of the HR index for the phenotype in A. The HR score was performed in 6 biological replicates and is presented as dots in the figure, where the size of each dot represents the number of samples with that score. Figure 33C shows the results of q-PCR determination of replicon copy number. The raGFP copy number was determined by q-PCR 3 days after Agrobacterium infection in tobacco. The control group consisted of co-expressed empty vector (EV). Error bars represent the standard deviation between the results of 6 replicates (including 3 biological replicates and 2 technical replicates). A "****" indicates a highly significant difference (Tukey's test: p < 0.0001).

[0264] Figure 34: Schematic diagram of the NRC3 mutant library.

[0265] Figure 35: Attenuation analysis of NRC3 screening experiment.

[0266] Figure 35a shows the screening pattern using NRC3 variants. Figure 35b shows the screening pattern using NRC3 variants. D480V The distribution of variant decay indices for template selection. The averaged decay indices of the four barcodes for the same mutation are plotted as a beehive plot. The vertical axis represents the logarithm of the average decay index of each variant.

[0267] Figure 36: NRC3 D480V To verify mutation sites using templates.

[0268] Figure 36A shows the results of tobacco fluorescence observation. Using NRC3... D480V Generate NRC3 for the template D480VIn the screening experiment, the top ten mutation sites in the decay score were combined. These ten protein variants were co-expressed with SS15 in tobacco. Autonomously replicating BeYDV-GFP replicons were co-expressed in tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated three times in parallel (bar = 1 cm). Figure 36B shows the statistical results of the tobacco HR index. The HR index was calculated 3 days after Agrobacterium infection in tobacco. The HR score was performed in six biological replicates and is presented as dots in the figure, where the size of each dot represents the number of samples with that score. The treatment group and the control group co-expressed SS15 and the empty vector (EV), respectively.

[0269] Figure 37: Transient expression results of NRC3 variants in tobacco.

[0270] According to NRC3 D480V The results of mutation site screening yielded nine variants using NRC3 as a template. These variants were transiently expressed individually in tobacco, with the control group consisting of the self-activating D480V mutation of NRC3. Autonomously replicating BeYDV-GFP replicons were co-expressed in tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated three times in parallel at a bar of 1 cm.

[0271] Figure 38: Verification of NRC3 variants using Pto / AVRPto, Gpa2 / RBP1, and Rx / CP, respectively.

[0272] Generate NRC3 template based on NRC3 D480V In the screening experiment, the top ten mutation sites with the highest decay scores were combined. These ten variants were co-expressed in tobacco with three groups of resistance / effective proteins and SS15. Autonomously replicating BeYDV-GFP replicons were co-expressed in tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated three times in parallel, bar = 1 cm. (B) Statistical results of the tobacco HR index. The HR index was calculated 3 days after Agrobacterium infection in tobacco. The HR score was performed in 6 biological replicates and is presented as dots in the figure, where the size of each dot represents the number of samples with that score. The treatment group and the control group co-expressed SS15 and the empty vector (EV), respectively.

[0273] Figure 39: Verification of NRC3 variants using AVRcap1b as an inhibitor.

[0274] Figure 39A shows the results of tobacco fluorescence observation. Using NRC3 as a template, E316P+N317K, E316K, and T315W mutations were generated. These three variants were co-expressed in wild-type tobacco with AVRcap1b and Pto / AVRPto or Gpa2 / RBP1, respectively. They were also co-expressed in nrc2 / 3 / 4 knockout tobacco with AVRcap1b and Rx / CP, respectively. Autonomously replicating BeYDV-GFP replicons were co-expressed in tobacco to characterize cell death. White light and fluorescence images were collected 3 days after Agrobacterium infection. The experiment was repeated three times in parallel, bar = 1 cm. Figure 39B shows the statistical results of the tobacco HR index. The HR index was calculated 3 days after Agrobacterium infection, with HR scores performed in 6 biological replicates. The results are presented as dots in the figure, where the size of each dot represents the number of samples with that score. The treatment and control groups co-expressed AVRcap1b and the empty vector (EV), respectively.

[0275] Figure 40: In vitro interaction results between SS15 and NRC3. The interaction between the NRC3 variant and SS15 was revealed by a yeast two-hybrid assay. Transformed yeast cells were spotted on plates with specific amino acid deficiencies, and yeast single clones were photographed 3 days after spotting. SD was a synthetic medium, and AbA was a cyclic peptide antibiotic.

[0276] Figure 41: Results of the interaction between SS15 and NRC3 in tobacco.

[0277] Figure 41A shows the qualitative detection results of luciferin. Figure 41B shows the quantitative detection results of luciferin. Co-expressed REN was used as an internal control. Luciferin was qualitatively and quantitatively detected 3 days after Agrobacterium infection of tobacco. Figure 41C shows the qualitative detection results of betaine. Figure 41D shows the quantitative detection results of betaine. Betaine was qualitatively detected and quantitatively measured using a 550 nm absorption wavelength 5 days after Agrobacterium infection of tobacco. Wild-type NRC3 (NRC3wt) and its variant were fused with the GAL4 binding domain, and SS15 was fused with the transcriptional activation domain VP64. These two fusion vectors were co-expressed in tobacco with the LUC / RUBY reporter vector. UAS-LUC / RUBY served as a negative control, and co-expression of the GAL4-VP64 fusion protein with UAS-LUC / RUBY served as a positive control. The presence of "****" indicates a highly significant difference (Tukey's test: p < 0.0001). The presence of "ns" indicates no significant difference (Tukey's test: p>0.05).

[0278] Figure 42: Results of single colony counting with DC3000.

[0279] Wild-type NRC3 (WT) and its variants were co-expressed with Pto in tobacco. The treatment group and control group co-expressed the empty vector (EV) (Figure 42A) and SS15 (Figure 42B), respectively. Three days after Agrobacterium infection of tobacco, the injection area was inoculated with RFP-tagged DC3000 strain. Three days after DC3000 inoculation, the number of DC3000 single colonies was counted. The presence of "****" indicates a highly significant difference (Tukey's test: p < 0.0001), while the presence of "ns" indicates no significant difference (Tukey's test: p > 0.05). Detailed Implementation

[0280] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0281] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0282] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0283] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0284] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0285] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0286] In this instruction manual, when "room temperature" or "room temperature" is used, the temperature can be 15-25℃.

[0287] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0288] The term "amino acid" in this invention refers to the basic building block of proteins in which an amino and a carboxyl group are bonded to the same carbon atom. Exemplarily, an amino acid is selected from one or more of the following: glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, proline, hydroxyproline, 5-aminolevulinic acid, or derivatives of any of the above-mentioned amino acids. Furthermore, the amino acid may also be other types of amino acids in the art.

[0289] According to the present invention, the three-letter codes and single-letter codes for amino acids used are as described in J. biol. chem, 243, p3558 (1968). The amino acids and their abbreviations and English abbreviations in this invention are as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine ​​(Cys, C); lysine (Lys, K); proline (Pro, P).

[0290] As used herein, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a single fragment or as a component of a larger nucleotide sequence structure derived from a nucleotide sequence isolated at least once in number or concentration, capable of being recognized, manipulated, and recovered using standard molecular biology methods (e.g., using cloning vectors). This also includes an RNA sequence (i.e., A, U, G, C) when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), where "U" replaces "T". In other words, "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (single fragments or entire fragments), or it can be a component or part of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

[0291] When the terms "comprising" or "including" are used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the stated sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still possessing the activities described in this invention. Furthermore, those skilled in the art will understand that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in certain practical situations (e.g., when expressed in a specific expression system) without substantially affecting the polypeptide's function. Therefore, when describing a specific polypeptide amino acid sequence in this application specification and claims, although it may not contain the methionine encoded by the start codon at the N-terminus, the sequence containing that methionine is still covered, and correspondingly, its encoding nucleotide sequence may also contain the start codon; and vice versa.

[0292] As used herein, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. As used herein, "naturally occurring" and "wild-type" are synonyms.

[0293] As used herein, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0294] As used in this invention, the term "mutated amino acid" includes "one or more amino acids that have been substituted, repeated, deleted, or added." In this invention, the term "mutation" refers to a change in the amino acid sequence.

[0295] In some embodiments, the "mutation" of this invention may be selected from "conservative mutations." In this invention, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0296] As used in this invention, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0297] As used herein, the terms "sequence identity" and "identity percentage" refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotide or polypeptide and scoring the number of positions in the aligned polynucleotide or polypeptide containing the same nucleotide or amino acid residues, comparing this to the number of positions in the aligned polynucleotide or polypeptide containing different nucleotide or amino acid residues. Polynucleotides may differ at a position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or deletions in one or two polynucleotides). Polypeptides may differ at a position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleted amino acids (i.e., amino acid insertions or deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of amino acid residues in the polynucleotide or polypeptide. For example, the percentage of identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide and then multiplying by 100.

[0298] In some embodiments, when comparing and aligning two or more sequences or subsequences using sequence comparison algorithms or by visual inspection to measure maximum correspondence, the two or more sequences or subsequences have a “sequence identity” or “percentage of identity” of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of nucleotides. In some embodiments, the sequences are substantially identical along the entire length of any one or two compared biopolymers (e.g., polynucleotides).

[0299] As used herein, the terms "corresponding" or "corresponding" have the meaning commonly understood by one of ordinary skill in the art. Specifically, "corresponding" means that, after homology or sequence identity alignment, one sequence corresponds to a specified position in another sequence. Thus, for example, regarding "corresponding to the 150th amino acid residue of the amino acid sequence shown in Sequence 1," if a 6×His tag is added to one end of the amino acid sequence shown in Sequence 1, then the 150th position in the resulting mutant corresponding to the 156th position of the amino acid sequence shown in Sequence 1 could be the 156th position.

[0300] As used in this invention, the term "expression" includes any step involving RNA and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0301] In this specification, the terms “nucleic acid construct,” “construct,” or “construct” refer to a polynucleotide that encodes a polypeptide or domain or module effectively linked to a suitable regulatory sequence that is essential for polynucleotide expression in selected cells or strains.

[0302] As used in this invention, the terms "vector" and "expression vector" refer to vectors, such as recombinant vectors, suitable for expressing nucleotide sequences of interest in plants.

[0303] In this specification, "introducing" a nucleic acid molecule (e.g., a vector containing a controlled artificial replicon of a geminivirus or a component of a plant directed evolution / screening system) into a plant cell means presenting the nucleic acid molecule to the plant cell so that the nucleic acid molecule enters the interior of the plant cell.

[0304] Invention Details

[0305] <Controlled artificial replicon of twin viruses>

[0306] In some aspects of the invention, a geminivirus-controlled artificial replicon is provided, comprising two long intergenic regions (LIRs) derived from common soybean yellow dwarf virus (BeYDV), with the two LIRs located on opposite sides of the artificial replicon, and optionally, the target gene to be replicated is contained between the two LIRs.

[0307] In some embodiments, the nucleotide sequence of the LIR is selected from at least one of the following groups (i)-(iv):

[0308] (i) Contains a nucleotide sequence as shown in SEQ ID NO:1;

[0309] (ii) A mutant sequence of the nucleotide sequence shown in SEQ ID NO:1, wherein the mutant sequence has a mutated nucleotide at one or more positions corresponding to the sequence shown in SEQ ID NO:1, and the mutant sequence has the function or activity of the nucleotide sequence shown in SEQ ID NO:1.

[0310] (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii) and has the function or activity of the nucleotide sequence shown in SEQ ID NO:1;

[0311] (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in SEQ ID NO:1.

[0312] In some specific implementations, the nucleotide sequence of the LIR is shown in SEQ ID NO:1.

[0313] In some alternative implementations, a small intergenic region (SIR) is included between the two LIRs.

[0314] In some specific implementations, the nucleotide sequence of the SIR is shown in SEQ ID NO:2.

[0315] In some preferred embodiments, no SIR is included between the two LIRs.

[0316] In some exemplary embodiments, the structure of the controlled artificial replicon of a geminivirus is as follows:

[0317] [LIR]-[Target gene to be replicated]-[LIR].

[0318] In some implementations, the target gene is operatively linked to an expression regulatory sequence.

[0319] In this specification, the terms "expression regulatory sequence" and "expression regulatory element" are used interchangeably, referring to a nucleotide sequence located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence and affecting the transcription, RNA processing, or stability or translation of the relevant coding sequence. Plant expression regulatory elements refer to nucleotide sequences capable of controlling the transcription, RNA processing, or stability or translation of a nucleotide sequence of interest in plants. Expression regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences. A "promoter" refers to a nucleic acid fragment capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the invention, a promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from a plant cell. A promoter may be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0320] In one specific embodiment, the target gene to be replicated in the controlled artificial replicon of the geminivirus is a coding sequence for a fluorescent protein. In this invention, when the target gene to be replicated is a coding sequence for a fluorescent protein, the controlled artificial replicon of the geminivirus is also referred to as a reporter replicon.

[0321] In some more specific implementations, an artificial intron is inserted into the coding sequence of the fluorescent protein. The N-terminal coding sequence of the fluorescent protein and the 5' end of the artificial intron are constructed at the posterior end of the replicon, while the 3' end of the artificial intron and the C-terminal coding sequence of the fluorescent protein are constructed at the anterior end of the replicon. When rolling circle replication does not occur in the controlled artificial replicon of the geminivirus, the fluorescent protein cannot form a complete coding frame and therefore cannot report fluorescence. However, when rolling circle replication occurs, the two LIRs merge, and the presence of the artificial intron causes the LIR to be recognized as a single intron and spliced ​​out post-transcriptionally. The N-terminal and C-terminal coding sequences of the fluorescent protein merge into a complete coding sequence, thus reporting fluorescence.

[0322] In some exemplary embodiments, the report replicon is structured as follows (report replicon 5' end (front end) to 3' end (back end)):

[0323] [LIR]-[3' end of the artificial intron]-[C-terminal coding sequence of the fluorescent protein]-[Terminator]-[SIR]-[Promoter]-[N-terminal coding sequence of the fluorescent protein]-[5' end of the artificial intron]-[LIR]

[0324] In this invention, there are no particular restrictions on the types of fluorescent proteins. For example, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc., can be used, but it is not limited to these.

[0325] In some implementations, the replication of the artificial replicon is controlled by Rep and / or RepA proteins derived from common bean yellow dwarf virus (BeYDV).

[0326] In some implementations, the replication of the artificial replicon is controlled by the Rep protein derived from common bean dwarf virus (BeYDV).

[0327] In some preferred embodiments, the replication of the artificial replicon is controlled by the Rep and RepA proteins derived from common bean yellow dwarf virus (BeYDV).

[0328] In some specific implementations, in plant cells that have been introduced with a controlled artificial replicon of geminiviruses (e.g., a vector containing the controlled artificial replicon of geminiviruses), the replication of the controlled artificial replicon of geminiviruses, or its replication at a high or low level, is controlled by introducing or not introducing Rep and / or RepA proteins (e.g., a vector expressing Rep and / or RepA proteins).

[0329] In some specific embodiments, the amino acid sequence of the Rep protein is selected from at least one of the following groups (i)-(iv):

[0330] (i) Contains an amino acid sequence as shown in SEQ ID NO:3;

[0331] (ii) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:3;

[0332] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:3, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:3.

[0333] (iv) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:3 under stringent conditions, and the amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:3, wherein the stringent conditions are moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0334] In some specific embodiments, the amino acid sequence of the RepA protein is selected from at least one of the following groups (i)-(iv):

[0335] (i) Contains an amino acid sequence as shown in SEQ ID NO:4;

[0336] (ii) An amino acid sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:4;

[0337] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:4, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:4.

[0338] (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:4 under stringent conditions, and said amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:4, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

[0339] In some specific implementations, the amino acid sequence of the Rep is shown in SEQ ID NO:3.

[0340] In some specific implementations, the amino acid sequence of RepA is shown in SEQ ID NO:4.

[0341] <Directed Evolution / Selection System in Plants>

[0342] In some aspects of the invention, a plant-based directed evolution / screening system is provided for directed evolution or screening of genetic elements to obtain mutants of the genetic elements having desired functions.

[0343] In some embodiments, the plant-based directed evolution / screening system includes:

[0344] (i) Controlled artificial replicons of twin viruses as described in the section on <Controlled Artificial Replicons of Gemini Viruses>;

[0345] (ii) Rep and / or RepA proteins as described in the section on <Controlled Artificial Replicons of Geminiviruses>, and

[0346] (iii) Mutants or mutant libraries of genetic elements;

[0347] The replication of the controlled artificial replicon of the twin virus is configured to be associated with the desired function of the mutant of the genetic element.

[0348] (Genetic elements)

[0349] As used herein, the term "genetic element" refers to a nucleotide sequence / nucleic acid molecule that can perform a specific function within a cell, preferably within plant cells. Examples of genetic elements include, but are not limited to, protein-coding sequences, functional RNA (e.g., tRNA, siRNA, etc.) coding sequences, and expression regulatory sequences such as promoter sequences, enhancer sequences, and terminator sequences.

[0350] In some preferred embodiments, the genetic element is derived from a plant or is intended to be applied to a plant.

[0351] (library)

[0352] In the context of this specification, the term "library" is used in its known meaning in the fields of cell biology and molecular biology, referring to a collection of different nucleic acid fragments / molecules. One particular type of library is a library containing random mutants generated through random mutagenesis. Another example is a designed (or synthesized) library containing specially engineered different nucleic acid fragments / molecules.

[0353] In some implementations, the mutant library of the genetic element is obtained by inserting the coding sequences of multiple mutants of the genetic element into a vector, respectively.

[0354] In some implementations, multiple mutants of the genetic element are generated through random mutagenesis.

[0355] In some implementations, the library can be generated by randomly mutagenesis of the genetic elements in the vector.

[0356] In the context of this specification, the term "random mutagenesis" is used in its known meaning in the fields of cell biology and molecular biology; it refers to a method in which DNA mutations are randomly introduced to produce mutant genes and proteins. Many of these mutant genes can then be compiled into a library. Non-limiting examples of random mutagenesis methods include error-prone PCR, UV radiation, and chemical mutagens.

[0357] (Plants and plant cells)

[0358] In some implementations, the replication of the controlled artificial replicon of the geminivirus refers to the replication of the controlled artificial replicon of the geminivirus in plant cells.

[0359] In some implementations, the plant-based directed evolution / screening system is used within plant cells.

[0360] The plants described in this invention can be monocotyledonous or dicotyledonous plants, as long as the controlled artificial replicon of the betavirus can replicate in its cells. Suitable plants include, but are not limited to, corn, wheat, rice, barley, sorghum, beans, beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, and tobacco.

[0361] In some embodiments, the plant cells are isolated plant cells. In some embodiments, the plant cells are protoplast cells.

[0362] In some preferred embodiments, the plant cells are cells within plant tissues, organs, or bodies; that is, the cells are not isolated from plant tissues, organs, or bodies. For example, the plant cells may be leaf cells.

[0363] (Carrier)

[0364] In some implementations, the coding sequences of the controlled artificial replicon of the geminivirus, the Rep and / or RepA proteins, and the coding sequences of mutants of genetic elements or mutants in mutant libraries can be constructed into different vectors, or any two or more can be constructed into the same vector.

[0365] In some embodiments, the vector is circular DNA, such as double-stranded or single-stranded circular DNA. In some embodiments, the vector is a plasmid. In some embodiments, the vector is microcircular DNA.

[0366] In some embodiments, the vector is an Agrobacterium vector, such as, but not limited to, vectors of the pBin, pCB301, pEAQ, pCambia, pLSU, pGD, pGreenII, pLX, pRi series, or vectors obtained by modification based on the above vectors.

[0367] In some preferred embodiments, the vector is the pPhi vector. This is a smaller Agrobacterium vector system more suitable for Agrobacterium infiltration transformation, which is beneficial for the directed evolution and screening of the plant in vivo directed evolution / screening system provided by this invention.

[0368] An example pPhi vector sequence is as follows:

[0369] pPhi-35Sp-GFP (SEQ ID NO:208)

[0370] in:

[0371] Uppercase letters and single underscores indicate sequences of type RB;

[0372] Uppercase letters and double underscores indicate sequences where the value is LB;

[0373] The lowercase letters and double underscores represent the sequence of the kanamycin resistance gene;

[0374] The italicized and bolded portions are sequences of the replication initiation sites of the pUC vector;

[0375] The gray background represents the sequence of the pVS1 oriV replication origin site of the pVS1 replication system;

[0376] The lowercase letters and single underscores represent the nucleotide sequence encoding GFP.

[0377] SEQ ID NO:208 is an exemplary vector sequence of GFP, where the nucleotide sequence of the target gene to be expressed between RB and LB is the GFP expression cassette. Those skilled in the art can replace the part before RB and LB with the expression cassette of the target gene to be expressed, depending on the target gene to be expressed.

[0378] Therefore, in some embodiments, such as the expression cassette between RB and LB of the expression vector shown in SEQ ID NO:208, the expression cassette of the target gene to be expressed can be replaced with the expression cassette of the target gene to be expressed.

[0379] The Agrobacterium vector system containing the pPhi vector also includes an auxiliary plasmid with the following nucleotide sequence: pSRK2 (SEQ ID NO:209).

[0380] in:

[0381] The underlined portion represents the sequence from the oriV replication origin site of the pRK2 replication system;

[0382] The single underlined portion is the sequence encoding trfA, a replication initiation protein from the pRK2 replication system;

[0383] The double-underlined portion represents the sequence of the spectinomycin resistance gene;

[0384] The italicized and bold text indicates the sequence of the replication start site of the pUC vector;

[0385] The uppercase letters represent the nucleotide sequence encoding pVS1 StaA, a stable protein from the pVS1 replication system;

[0386] The gray background represents the nucleotide sequence encoding the replication initiation protein pVS1 RepA from the pVS1 replication system.

[0387] This invention creatively discovers that the pPhi carrier has the following advantages:

[0388] First, the pPhi vector can maintain high copy numbers in both Escherichia coli and Agrobacterium.

[0389] Secondly, the pPhi vector has a high plant transformation efficiency, which is on par with or higher than that of the currently most efficient pCambia vector.

[0390] Furthermore, because the pPhi vector has a very small skeleton, it is very easy and flexible to construct, making it very suitable for library construction.

[0391] Finally, due to its small size and high construction efficiency, it can skip the transformation of E. coli and directly transform Agrobacterium, with a high positive rate, thus shortening the entire experimental cycle and reducing the workload.

[0392] In some exemplary embodiments, the mutant library of the genetic element is obtained by inserting multiple mutants of the genetic element into a controlled artificial replicon of geminivirus, i.e., the vector containing the controlled artificial replicon of geminivirus also contains the coding sequence of the mutant of the genetic element or a mutant from the mutant library. In other words, the target gene to be replicated in the controlled artificial replicon of geminivirus includes the coding sequence of the mutant of the genetic element or a mutant from the mutant library. In such embodiments, the target genes to be replicated in multiple controlled artificial replicons of geminivirus each contain multiple mutants of the genetic element, forming a library of mutants of the genetic element, whereby the mutant is amplified when the controlled artificial replicon of geminivirus replicates.

[0393] In some exemplary embodiments, the vector containing a controlled artificial replicon of a geminivirus does not contain the coding sequence of a mutant of the genetic element or a mutant in a mutant library. Therefore, the coding sequence of a mutant of the genetic element or a mutant in a mutant library can be provided by constructing it into another vector to form a mutant library.

[0394] For example, in some exemplary embodiments, the vector expressing Rep and / or RepA proteins may contain coding sequences of mutants of genetic elements or mutants from a mutant library. As another example, in some exemplary embodiments, coding sequences of mutants of genetic elements or mutants from a mutant library may be constructed into a separate vector that does not contain coding sequences for the controlled artificial replicon of geminiviruses and / or Rep and / or RepA proteins.

[0395] In some exemplary embodiments, the vector containing a controlled artificial replicon of a geminivirus contains coding sequences for Rep and / or RepA proteins. The expression cassette for Rep and / or RepA proteins typically contains coding nucleotide sequences for Rep and / or RepA proteins and expression regulatory elements operatively linked thereto.

[0396] In some exemplary embodiments, the vector containing a controlled artificial replicon of a geminivirus does not contain the coding sequence for the Rep and / or RepA proteins. Therefore, the Rep and / or RepA proteins need to be provided in the trans form. Vectors for expressing the Rep and / or RepA proteins typically contain expression cassettes for the Rep and / or RepA proteins.

[0397] In some exemplary embodiments, the plant cell already contains a vector for expressing Rep and / or RepA proteins, and / or the plant cell genome has an expression cassette for Rep and / or RepA proteins integrated into it.

[0398] (The replication of the controlled artificial replicon of the geminivirus is associated with the desired function of the mutant of the genetic element.)

[0399] In some implementations, “the replication of the controlled artificial replicon of the geminivirus is configured to be associated with the desired function of a mutant of the genetic element” includes setting the replication level of the controlled artificial replicon of the geminivirus to be associated with the desired function of a mutant of the genetic element.

[0400] The "replication level" of a controlled artificial replicon of geminiviruses can be determined by detecting the copy number of the controlled artificial replicon. Methods for detecting the copy number of a controlled artificial replicon of geminiviruses are known in the art, including but not limited to PCR (e.g., quantitative real-time PCR) or sequencing (e.g., deep sequencing). In some embodiments, when the controlled artificial replicon of geminiviruses includes a fluorescent protein-coding sequence (e.g., in the case of a reporting replicon), the replication level of the controlled artificial replicon of geminiviruses can also be determined by detecting the fluorescence intensity produced by fluorescent protein expression.

[0401] In some specific implementations, the controlled artificial replicon of the geminivirus is replicated (amplified) via rolling circle replication.

[0402] In this invention, the replication level of controlled artificial replicons of plant cell geminiviruses (including the activity or expression level of Rep and / or RepA proteins of plant cell geminiviruses) can be directly or indirectly associated with the desired function of the genetic element mutant. Those skilled in the art can achieve such an association based on the type of genetic element and the specific desired function of its mutant.

[0403] The replication level of the controlled artificial replicon of the geminivirus was set to be associated with the desired function of the mutant of the genetic element.

[0404] positive association

[0405] In some implementations, "the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element" includes a positive association (coupling), specifically including: such that the replication level of the controlled artificial replicon of the geminivirus with the desired function in the genetic element mutant is higher, preferably significantly higher, than the replication level of the controlled artificial replicon of the geminivirus without the desired function in the genetic element mutant; or, in plant cells with the genetic element mutant having the desired function, the replication level of the controlled artificial replicon of the geminivirus (e.g., reporter replicon) is higher, preferably significantly higher, than in plant cells with the genetic element mutant without the desired function.

[0406] For example, a mutant of a genetic element having the desired function can cause the replication of a controlled artificial replicon of a geminivirus in plant cells, while a variant of a genetic element without the desired function causes the controlled artificial replicon of a geminivirus to not replicate in plant cells; or preferably, a mutant of a genetic element having the desired function can cause high-level replication of a controlled artificial replicon of a geminivirus in plant cells, while a variant of a genetic element without the desired function causes low-level replication or no replication of a controlled artificial replicon of a geminivirus in plant cells. When the controlled artificial replicon of a geminivirus containing a mutant of a genetic element with the desired function is amplified or significantly amplified relative to other controlled artificial replicons of a geminivirus containing a mutant of a genetic element with the desired function due to replication or high-level replication, enrichment of the mutant of a genetic element with the desired function can be achieved; or, in plant cells containing a mutant of a genetic element with the desired function, differences in replication or replication levels of the controlled artificial replicon of a geminivirus can be detected, for example, by producing fluorescence, or producing stronger fluorescence intensity, or more copy numbers, relative to plant cells without the mutant of a genetic element with the desired function.

[0407] Rep and / or RepA proteins are replication initiation proteins of geminiviruses, and their activity or expression levels are generally positively correlated within a certain range with the replication level (e.g., copy number) of the controlled artificial replicon of geminiviruses. Therefore, in some embodiments, "the replication level of the controlled artificial replicon of geminiviruses is set to be associated with the desired function of a mutant of the genetic element" also includes setting the activity or expression level of Rep and / or RepA proteins in the plant cells to be associated with the desired function of the mutant of the genetic element. For example, the activity or expression level of Rep and / or RepA proteins in plant cells containing mutants of genetic elements having the desired function may be higher, preferably significantly higher, than the activity or expression level of Rep and / or RepA proteins in plant cells containing mutants of genetic elements not having the desired function. In some embodiments, the activity of the Rep and / or RepA proteins is the activity mediating (initiating) the replication of the controlled artificial replicon of geminiviruses, which can be determined, for example, by detecting the replication level of the controlled artificial replicon of geminiviruses. For example, a mutant of a genetic element having the desired function can be made to result in the expression of Rep and / or RepA proteins in plant cells, while a mutant of a genetic element not having the desired function can result in the non-expression of Rep and / or RepA proteins in plant cells; or a mutant of a genetic element having the desired function can be made to result in high-level expression of Rep and / or RepA proteins in plant cells, while a mutant of a genetic element not having the desired function can result in low-level expression or non-expression of Rep and / or RepA proteins in plant cells. Alternatively, a mutant of a genetic element having the desired function can be made to result in the activity of Rep and / or RepA proteins in plant cells, while a mutant of a genetic element not having the desired function can result in the inactivity of Rep and / or RepA proteins in plant cells; or a mutant of a genetic element having the desired function can result in high activity of Rep and / or RepA proteins in plant cells, while a mutant of a genetic element not having the desired function can result in low activity or inactivity of Rep and / or RepA proteins in plant cells. The expression or activity, or high level of expression or activity, of Rep and / or RepA proteins in the plant cells will lead to the amplification or significant amplification of the controlled artificial replicon of the geminivirus, thereby achieving the enrichment of genetic element mutants with the desired function, or the detection of differences in replication or replication levels.

[0408] The terms "low level" or "low activity" used in this article are relative to "high level" or "high activity" and do not necessarily mean that the activity is below the normal level or normal activity.

[0409] In the context of this specification, the term “expression level” is used in its known meaning in the fields of cell biology and molecular biology; it refers to the transcriptional and / or translational levels of DNA fragments and their derived mRNAs, respectively.

[0410] In some implementations, a mutant library of genetic elements (containing multiple mutants of the genetic elements) can be transformed into a population of plant cells (e.g., leaves), the population of plant cells can be cultured, and the genetic element mutants enriched in the population of plant cells can be detected and selected, thereby achieving directed evolution of the genetic elements.

[0411] In some specific embodiments, the directed evolution of genetic elements is accomplished by coupling the expression or activity of Rep and / or RepA proteins in the plant cells with the desired function of the mutant of the genetic element. In some embodiments, the directed evolution of genetic elements is accomplished by: functional genetic elements activating (or highly expressing) Rep and / or RepA proteins, thereby driving rolling circle replication and achieving self-enrichment; and non-functional genetic elements failing to activate (or expressing at low levels) Rep and / or RepA protein expression, thus failing to achieve enrichment.

[0412] For example, when the genetic element is an expression regulatory element (such as a promoter, enhancer, etc.), the coding sequence of the Rep and / or RepA proteins can be directly placed under the control of the expression regulatory element mutant (such as a promoter mutant, enhancer mutant, etc.). If the mutant can enhance gene expression, it can lead to increased expression of the Rep and / or RepA proteins, which in turn leads to increased replication of the geminivirus controlled artificial replicon and the corresponding expression regulatory element mutant (such as a promoter mutant). By detecting significantly enriched mutant sequences, expression regulatory element mutants that enhance gene expression can be obtained, i.e., evolved expression regulatory elements.

[0413] When the genetic element is a protein-coding sequence, the intended function of the protein it encodes can be associated with the activity or expression level of the Rep and / or RepA proteins.

[0414] As demonstrated in the examples, the plant-based directed evolution / screening system provided by the present invention can complete 10 [evolutions / screenings] in a single tobacco leaf when used for directed evolution in plants. 5 The system can screen for multiple types of targeted gene variants and complete a round of directed evolution within 4 days.

[0415] negative association

[0416] In other embodiments, "the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element" includes a negative association (coupling), specifically including: such that the replication level of the controlled artificial replicon of the geminivirus with the desired function in the genetic element mutant in plant cells is lower, preferably significantly lower, than the replication level of the controlled artificial replicon of the geminivirus without the desired function in plant cells. Alternatively, the replication level of the controlled artificial replicon of the geminivirus (e.g., reporter replicon) in plant cells with the genetic element mutant having the desired function is lower, preferably significantly lower, than the replication level of the controlled artificial replicon of the geminivirus (e.g., reporter replicon) in plant cells with the genetic element mutant not having the desired function.

[0417] In some specific embodiments, the controlled artificial replicon of a geminivirus containing a mutant of a genetic element with the desired function, due to the desired function produced by replication (e.g., generating immune function, leading to, for example, cell death infected by a pathogen), causes its replication level in plant cells, as determined by copy number, to be lower than that of a controlled artificial replicon of a geminivirus without the desired function in plant cells, i.e., negative directed evolution.

[0418] In some specific implementations, non-replication or low-level replication of the controlled artificial replicon of the geminivirus can be detected in plant cells containing the genetic element mutant with the desired function, relative to plant cells without the genetic element mutant with the desired function; for example, weaker fluorescence intensity or fewer copy number.

[0419] Similarly, the aforementioned negative correlation is also suitable for setting the activity or expression level of Rep and / or RepA proteins in plant cells to be associated with the desired function of the genetic element mutant. For example, the activity or expression level of Rep and / or RepA proteins in plant cells containing genetic element mutants having the desired function can be lower, preferably significantly lower, than the activity or expression level of Rep and / or RepA proteins in plant cells containing genetic element mutants not having the desired function.

[0420] (Exemplary directed evolution system in plants - forward directed evolution)

[0421] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of a mutant of the genetic element” includes coupling antiviral tRNA with rolling circle replication, i.e., the genetic element includes antiviral tRNA.

[0422] In the context of this specification, the term "anti-viral tRNA" refers to tRNA that can promote the reading of stop codons and inhibit viral replication.

[0423] In some specific implementations, a stop codon and a self-cleaving peptide coding sequence are inserted at the 5' end of the Rep and / or RepA protein coding sequences. To avoid potential ribosomal leakage of Rep and / or RepA protein expression, a tandem initiation codon (TIC) is inserted before the stop codon. Thus, Rep and / or RepA proteins can only be expressed when the stop codon is read through.

[0424] In some specific implementations, plant-based directed evolution systems for the directed evolution of antiviral tRNAs include:

[0425] (i) a vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant from a mutant library, wherein the genetic element is an antiviral tRNA to be evolved; and,

[0426] (ii) A vector for expressing Rep and / or RepA proteins, wherein the expression cassette of Rep and / or RepA proteins contains: tandem initiation codons (TIC), a stop codon, a self-cleaving peptide coding sequence, and a Rep and / or RepA protein coding sequence.

[0427] In some specific implementations, the vector expressing Rep and / or RepA proteins contains nucleotide sequences as shown in any one of SEQ ID NO:90-92.

[0428] In plant-based directed evolution systems for the directed evolution of antiviral tRNAs, antiviral tRNAs that promote stop codon readout enable the stop codons in the expression cassettes of Rep and / or RepA proteins to be readout, thereby leading to the expression of Rep and / or RepA proteins, rolling circle replication, and ultimately the enrichment of functional antiviral tRNAs, thus achieving directed evolution.

[0429] (Exemplary directed evolution system in plants—negative directed evolution)

[0430] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of a mutant of the genetic element” includes coupling a mutant of an immune-related protein in the plant with rolling circle replication, i.e., the genetic element includes a mutant of an immune-related protein in the plant or its coding sequence, the desired function of which includes relieving the inhibition of proteins derived from pathogens.

[0431] In some specific implementations, a plant-based directed evolution / screening system for mutants of immune-related proteins in directed evolution plants includes:

[0432] (i) A vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant in a mutant library, wherein the genetic element is the coding sequence of a mutant of the immune-related protein to be evolved;

[0433] (ii) a vector expressing Rep and / or RepA proteins; and,

[0434] (iii) A vector expressing the pathogen-derived protein, which has an inhibitory effect on natural / initiated immune-related proteins.

[0435] In some embodiments, such as mutants of the immune-related protein having the function of relieving the inhibition of pathogen-derived proteins, when the mutant of the immune-related protein is activated (e.g., in the presence of pathogens or pathogen-derived proteins (e.g., pathogen effector proteins) in plant cells), the immune-related protein induces cell death (to confer resistance to the pathogen), thereby resulting in a reduction in the copy number of the controlled artificial replicon of the geminivirus.

[0436] In some specific implementations, the plant-based directed evolution / screening system for mutants of immune-related proteins in directed evolution plants also includes pathogens or pathogen-derived proteins (e.g., pathogen effector proteins) that activate plant cellular immune responses (e.g., cell death due to immune responses).

[0437] Those skilled in the art can select appropriate combinations based on known interactions between immune-related proteins and related proteins in pathogens, thereby employing the directed evolution or screening provided by the present invention to release the inhibition of specific pathogen-derived proteins of corresponding immune-related proteins.

[0438] In some optional embodiments, the immune-related proteins include proteins in the NLR network.

[0439] In some alternative implementations, the immune-related protein includes NRC.

[0440] In some exemplary embodiments, the immune-related protein includes NRC3, and the pathogen-derived protein includes SS15.

[0441] In some specific, exemplary embodiments, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling an NRC3 mutant with SS15 repression deactivated with rolling circle replication, i.e., the genetic element includes an NRC3 mutant with SS15 repression deactivated or its coding sequence.

[0442] In some specific implementations, the plant-based directed evolution system for directed evolution of NRC3 mutants that relieve SS15 inhibition includes:

[0443] (i) A vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant in a mutant library, wherein the genetic element is the coding sequence of an NRC3 mutant to be evolved to relieve SS15 inhibition.

[0444] (ii) a vector expressing Rep and / or RepA proteins; and,

[0445] (iii) Vectors that express SS15.

[0446] In some preferred embodiments, the coding sequence of the NRC3 mutant to be evolved, which has been freed from SS15 inhibition, includes an intron sequence. In some more preferred embodiments, the intron sequence includes a DNA barcode.

[0447] In other implementations, multiple mutants of genetic elements contained in a library of mutant genetic elements can be transformed into different populations of plant cells (e.g., leaves), and the different populations of plant cells can be cultured separately. The replication of the controlled artificial replicon of the geminivirus can be detected, for example, the replication level, or, in the case of a reporter replicon, the expression intensity of a fluorescent protein, or, for example, the copy number of the controlled artificial replicon of the geminivirus, to screen for mutants of functional genetic elements, thus achieving the screening of genetic elements.

[0448] (Example plant in vivo screening system - positive association)

[0449] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of a mutant of the genetic element” includes coupling an intein to Rep and / or RepA proteins, i.e., the genetic element includes an intein or the coding sequence of an intein.

[0450] In the context of this specification, the term "intepid" refers to a class of peptide segments that, after translation, are capable of self-splicing and releasing their free components, while their flanking exopins are linked by peptide bonds to form a mature protein. This process is similar to intron splicing. Intepids can perform both cis and trans splicing, and are therefore often used for segmented expression of longer protein-coding sequences.

[0451] In some specific implementations, the Rep and / or RepA proteins are separated into N-terminus and C-terminus, and then fused to the N-terminus (N-inpeptide) and C-terminus (C-inpeptide) of the inpeptide to be screened, respectively. The functional and mutually recognizing N-inpeptides and C-inpeptides interact within the plant cell, linking their exopeptides, namely the Rep and / or RepA proteins, into a complete protein.

[0452] In some specific implementations, the in vivo plant screening system for screening peptides includes:

[0453] (i) A vector expressing a first fusion protein, the first fusion protein comprising the N-terminus of the inteptide to be screened, and the N-terminus of Rep and / or RepA protein (or the C-terminus of Rep and / or RepA protein);

[0454] (ii) a vector expressing a second fusion protein, the second fusion protein comprising the C-terminus of the inteptide to be screened, and the C-terminus of Rep and / or RepA protein (or the N-terminus of Rep and / or RepA protein); and,

[0455] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0456] In some implementations, there are no particular restrictions on the length of the N-terminus and C-terminus of the Rep and / or RepA proteins, as long as a complete Rep and / or RepA protein can be formed.

[0457] In some preferred embodiments, the Rep and / or RepA proteins are split into N-terminus and C-terminus at cysteine ​​183 (C183) and fused to the N-terminus and C-terminus of the integrin to be screened, respectively.

[0458] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling a sequence-specific protease to Rep and / or RepA proteins, i.e., the genetic element includes a sequence-specific protease or its coding sequence.

[0459] In the context of this specification, the term "sequence-specific protease" refers to a protease that recognizes and cleaves its corresponding peptide segment (typically 6-9 amino acids). Sequence-specific proteases can be used for the expression of multiple proteins within the same coding frame, or for constructing genetic pathways to regulate the expression level of a specific gene.

[0460] In some specific implementations, a degron is fused to the N-terminus of the Rep and / or RepA proteins, and the Rep and / or RepA proteins are linked to the degron with a peptide that can be recognized by a specific protease to be screened. The presence of the degron can significantly reduce the half-life of the fused protein, causing it to be rapidly degraded by the host after translation. Since the Rep and / or RepA proteins play a crucial role in rolling circle replication, their half-life is naturally closely related to the level of rolling circle replication.

[0461] In some specific implementations, in vivo plant screening systems for screening sequence-specific proteases include:

[0462] (i) A vector expressing the sequence-specific protease to be screened;

[0463] (ii) a vector for expressing a fusion protein, said fusion protein comprising, from N-terminus to C-terminus: a degrader, a peptide recognized by the sequence-specific protease to be screened, and Rep and / or RepA protein; and

[0464] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0465] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of a mutant of the genetic element” includes coupling a sequence-specific recombinase to the Rep and / or RepA protein, i.e., the genetic element includes a sequence-specific recombinase or its coding sequence.

[0466] In the context of this specification, the term "recombinase" refers to an enzyme involved in the localized recombination process. It is responsible for recognizing and cleaving specific recombination sites and ligating two molecules involved in the recombination. "Sequence-specific recombinases" can recognize their corresponding DNA sequences and recombine the two DNA sequences. This recombination process can be reversible or irreversible. Recombinases have a wide range of applications, including vector construction, site-directed gene deletion, site-directed gene insertion, and chromosome manipulation.

[0467] In some more specific implementations, in the controlled artificial replicon of the geminivirus, two recombinase recognition sites are inserted at the 5' end of the coding sequence of the Rep and / or RepA proteins, and a terminator and a stop codon are inserted between the two recombinase recognition sites. When the recombinase is absent, both transcription and translation of the Rep and / or RepA proteins terminate between the two recombinase recognition sites; when the recombinase is present and functional, recombination occurs at the two recombinase recognition sites, the terminator and stop codon are removed, the Rep and / or RepA proteins are expressed normally, and rolling circle replication is activated.

[0468] In some more specific implementations, the in vivo plant screening system for screening sequence-specific recombinases includes:

[0469] (i) A vector expressing Rep and / or RepA proteins, the vector comprising a Rep and / or RepA protein coding sequence and two recombinase recognition sites located at the 5' end of the Rep and / or RepA protein coding sequence, and comprising a terminator and a stop codon between the two recombinase recognition sites;

[0470] (ii) A vector expressing the sequence-specific recombinase to be screened, and,

[0471] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0472] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling a self-cleavage peptide to Rep and / or RepA proteins, i.e., the genetic element includes the self-cleavage peptide or its coding sequence.

[0473] In the context of this specification, the term "self-cleaving peptide" refers to a class of short peptides that function similarly to protein hydrolases. When the ribosome translates to the glycine residue at the C-terminus of the self-cleaving peptide, a hydrolysis reaction occurs, releasing the self-cleaving peptide and its upstream proteins. However, the ribosome does not disintegrate; instead, translation continues at the proline residue at the C-terminus of the self-cleaving peptide. Self-cleaving peptides can be used for the equal expression of multiple genes within the same coding frame.

[0474] In some specific implementations, a degrader is fused to the N-terminus of the Rep and / or RepA proteins, and a self-cleaving peptide is used to link the Rep and / or RepA proteins to the degrader. Thus, the active self-cleaving peptide can cleave the degrader, thereby stabilizing the Rep and / or RepA proteins and activating the rolling circle replication of the replicon.

[0475] In some specific implementations, the in vivo plant screening system for screening self-cleaving peptides includes:

[0476] (i) a vector for expressing a fusion protein, wherein the fusion protein comprises, from the N-terminus to the C-terminus: a degrader, a self-cleaving peptide to be screened, and Rep and / or RepA protein; and,

[0477] (ii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0478] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling a transcriptional activator with Rep and / or RepA proteins, i.e., the genetic element includes a transcriptional activator or its coding sequence.

[0479] In the context of this specification, the term "transcription activator" is a DNA-binding protein capable of activating gene expression. Transcription activators regulate transcription by binding to upstream promoter elements. They are commonly used for gene expression regulation by recruiting endogenous factors that activate RNA polymerase activity to activate the transcription of target genes.

[0480] In some more specific embodiments, in a controlled artificial replicon of a geminivirus, a recognition sequence (e.g., a promoter) of a transcription activator is inserted upstream of the sequence encoding Rep and / or RepA proteins, and a DNA sequence or motif recognized by a known DNA-binding domain is inserted upstream of the recognition sequence of the transcription activator. The transcription activator is fused with the known DNA-binding domain, thereby recruiting the transcription activator upstream of the Rep and / or RepA proteins. The transcription activator, active in plants, can then initiate the expression of the Rep and / or RepA proteins, thereby activating rolling circle replication.

[0481] In some more specific implementations, the in vivo plant screening system for screening transcription activators includes:

[0482] (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by a known DNA-binding domain, a recognition sequence of the transcription activator to be screened, and a sequence encoding Rep and / or RepA proteins.

[0483] (ii) a vector expressing a fusion protein, said fusion protein comprising the selected transcription activator and the known DNA-binding domain, and,

[0484] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0485] In some preferred embodiments, the recognition sequence (e.g., promoter) of the transcription activator is either unrecognizable or barely recognized by the transcription activators in the plant cells in which the in vivo screening system for screening transcription activators is introduced.

[0486] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of a mutant of the genetic element” includes coupling a DNA-binding protein with Rep and / or RepA proteins, i.e., the genetic element is a DNA-binding protein or its coding sequence.

[0487] In the context of this specification, the terms "DNA-binding domain" or "DNA-binding protein" refer to proteins that can recognize and bind to specific DNA sequences or motifs, and are commonly used in fields such as transcriptional activation, transcriptional repression, and genome manipulation.

[0488] In some more specific embodiments, in a controlled artificial replicon of a geminivirus, a recognition sequence (e.g., a promoter) of a known transcription activator is inserted upstream of the sequence encoding Rep and / or RepA proteins, and a DNA sequence or motif recognized by a DNA-binding domain is inserted upstream of the recognition sequence of the known transcription activator. The known transcription activator is fused with the DNA-binding domain, such that the DNA-binding domain, capable of recognizing a specific DNA sequence or motif, can activate the expression of Rep and / or RepA proteins, thereby activating rolling circle replication.

[0489] In some more specific implementations, in vivo plant screening systems for screening DNA-binding proteins include:

[0490] (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by the DNA-binding domain to be screened, a recognition sequence of a known transcription activator, and a sequence encoding Rep and / or RepA proteins.

[0491] (ii) a vector for expressing a fusion protein, said fusion protein comprising the known transcription activator and the DNA-binding domain to be screened, and,

[0492] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0493] In some preferred embodiments, the recognition sequences (e.g., promoters) of known transcription activators are either unrecognized or barely recognized by the transcription activators in the plant cells in which the in vivo screening system for screening DNA-binding proteins is introduced.

[0494] (Exemplary plant in vivo screening system – reverse association)

[0495] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling a homologous recombination factor with rolling circle replication.

[0496] In the context of this specification, the term "DNA recombination factor" is a general concept referring to factors that function during DNA recombination in vivo. The efficiency of homologous recombination in plants has consistently been very low, a significant limiting factor in plant genome editing. Developing DNA recombination factors that can work efficiently in plants may solve this problem.

[0497] In some specific implementations, plant-based directed evolution systems for screening DNA recombination factors include:

[0498] (i) Vectors expressing DNA recombinant factors to be evolved;

[0499] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0500] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0501] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling DNA deaminase with rolling circle replication.

[0502] In the context of this specification, the term "DNA deaminase" refers to an enzyme that can deaminate cytosine and adenine, and has important applications in genome base editing.

[0503] In some specific implementations, plant-based directed evolution systems for screening DNA deaminases include:

[0504] (i) A vector expressing DNA deaminases to be evolved;

[0505] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0506] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0507] In some implementations, “the replication level of the controlled artificial replicon of the geminivirus is set to be associated with the desired function of the mutant of the genetic element” includes coupling the plant immunity elicitor with rolling circle replication.

[0508] In the context of this specification, plant immune elicitors can induce an immune response in plants, thereby conferring disease resistance.

[0509] In some specific implementation schemes, the plant-based directed evolution system for screening plant immune elicitors includes:

[0510] (i) Vectors expressing plant immune elicitors to be evolved;

[0511] (ii) A vector constitutively expressing Rep and / or RepA proteins;

[0512] (iii) A vector containing a controlled artificial replicon of geminiviruses, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of geminiviruses is detectable, for example, the target gene to be replicated is a fluorescent protein coding sequence or a DNA barcode. In some exemplary embodiments, the controlled artificial replicon of geminiviruses includes the reporter replicon described above.

[0513] It is understood that, based on the aforementioned plant in vivo directed evolution system, those skilled in the art can design corresponding plant in vivo screening systems, and similarly, based on the aforementioned plant in vivo screening system, those skilled in the art can design corresponding plant in vivo directed evolution systems. Furthermore, as mentioned above, the components of the aforementioned plant in vivo directed evolution system and plant in vivo screening system, such as at least one component, two components, or more, can be constructed in the same (same) vector, or separately constructed in different vectors.

[0514] <Directed Evolution / Screening Methods in Plants>

[0515] In some aspects of the present invention, a method for directed evolution / screening within plants is provided, the method employing a directed evolution / screening system as described in the section on directed evolution / screening systems within plants to perform directed evolution or screening of genetic elements to obtain mutants of the genetic elements having the desired function.

[0516] In some implementations, the plant-based directed evolution / screening method includes:

[0517] A population of plant cells transformed with the plant-based directed evolution / screening system (e.g., a controlled artificial replicon of a geminivirus, a mutant or mutant library of Rep and / or RepA proteins and genetic elements); and

[0518] A population of the plant cells is cultured.

[0519] The plants and plant cells described are as described in the section on "Directed Evolution / Screening Systems in Plants".

[0520] Plant-based directed evolution / screening systems (such as controlled artificial replicons of geminiviruses, Rep and / or RepA proteins, and mutants or mutant libraries of genetic elements) can be introduced into populations of plant cells using methods known in the art, such as protoplast transformation, callus transformation, Agrobacterium infiltration, and gene gun transformation.

[0521] In some preferred embodiments, plant-based directed evolution / screening systems (e.g., controlled artificial replicons of geminiviruses, Rep and / or RepA proteins, and mutants or mutant libraries of genetic elements) are introduced into a population of plant cells via Agrobacterium infiltration.

[0522] Agrobacterium infiltration refers to injecting Agrobacterium bacterial solution containing the target gene into plant leaves (e.g., leaves of Nicotiana benthamiana) using a syringe without the needle.

[0523] In some embodiments, the plant-based directed evolution / screening method further includes:

[0524] Detect and select mutants of genetic elements that have the desired function in the population of said plant cells.

[0525] In some specific embodiments, the detection and selection of genetic element mutants with the desired function in a population of plant cells can be performed using high-throughput sequencing. For example, total DNA can be extracted from the population of plant cells and high-throughput sequencing can be performed on the genetic elements. In other specific embodiments, the detection and selection of genetic element mutants with the desired function in a population of plant cells can also be performed by detecting and comparing fluorescence intensity.

[0526] In some embodiments, the plant-based directed evolution / screening method further includes:

[0527] Identify the function of the enriched genetic element mutants.

[0528] <Mutants of genetic elements or their coding sequences>

[0529] In some aspects of the invention, the invention provides genetic element mutants (or their coding sequences) obtained by employing the plant-based directed evolution / screening system described in the <Plant-based Directed Evolution / Screening System> section of the invention or by employing the plant-based directed evolution / screening method described in the <Plant-based Directed Evolution / Screening Method> section of the invention.

[0530] (Antiviral tRNA)

[0531] In some implementations, the genetic element mutant includes antiviral tRNA.

[0532] In some embodiments, the nucleotide sequence of the antiviral tRNA is selected from at least one of the following groups (i)-(iv):

[0533] (i) Contains a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153;

[0534] (ii) A mutant sequence of a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, wherein the mutant sequence has a mutated nucleotide at one or more positions of the sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, and the mutant sequence has the function or activity of the nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153;

[0535] (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153.

[0536] (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153, and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153.

[0537] In some specific implementations, the nucleotide sequence of the antiviral tRNA is shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153.

[0538] In some implementations, the virus is one whose replication and / or propagation are affected by stop codon readout.

[0539] In some exemplary embodiments, the virus includes viruses of the Virgaviridae and Tombusviridae families, which rely on stop codon readout to express SRS and RdRp, which are crucial for their replication.

[0540] In this invention, the Virgaviridae family comprises uncoated, rod-shaped, positive-stranded linear RNA viruses. Their genomes have a cap structure at the 5' end (5'-cap(m7GpppG)) and a tRNA-like structure at the 3' end. The viruses can be monodextrous, diploid, or tridid. This family currently includes over 60 species, divided into 7 genera: Goravirus, Furovirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus. The representative virus in the Virgaviridae family is Tobacco mosaic virus (TMV).

[0541] Fluticiviruses have a wide host range, including both monocotyledonous and dicotyledonous plants. They can be transmitted through mechanical friction, nematodes, insects, pollen, seeds, and other means.

[0542] Members of the Broomvirus family exhibit slightly different replication patterns; here, we take the genus *Tobacco Mosaic Virus*, which has the largest number of members, as an example. *Tobacco Mosaic Virus* encodes two replication proteins: the 126K protein is called the small replicase subunit (SRS), and the 183K protein is called the RNA-dependent RNA polymerase (RdRp). SRS and RdRp share the N-terminus, and RdRp is expressed through a leaked stop codon in the genome. In plant protoplasts, RdRp can initiate replication on its own, but in the plant body, both SRS and RdRp are indispensable.

[0543] The replication of tobacco mosaic virus can be divided into the following steps. After the virus invades plant cells, the viral particles first disintegrate, releasing the viral genomic RNA. Subsequently, viral genes are expressed, and the expressed RdRp and SRS bind to the 5' end of the viral genome, recruiting host factors to form the replication complex. Then, RdRp functions to synthesize the viral complementary strand. Finally, using the complementary strand as a template, progeny viral RNA is synthesized. The progeny RNA assembles with the viral capsid protein to form viral particles, which then carry out subsequent infection under the action of motility proteins.

[0544] In this invention, the family Tombusviridae comprises uncoated, spherical, positive-stranded linear RNA viruses. Their genomic RNA lacks a cap structure at the 5' end and also lacks polyadenylation or tRNA structures at the 3' end. However, the virus's 5' untranslated region possesses a 'Y'-shaped structure, which can serve as an internal ribosome entry site (IRES), enabling cap-independent translation initiation. The virus's 3' untranslated region contains a sequence that can interact with its 5' untranslated region over long distances, regulating viral replication, transcription, and translation. All viruses in this family are monodextrous. Based on genomic structure, Tombusviridae can be divided into 16 genera, with *Tombusvirus*, *Alphanecrovirus*, *Zeavirus*, and *Umbravirus* being representative genera. Tombusviridae viruses have a wide host range, infecting both monocotyledonous and dicotyledonous plants. These viruses can be transmitted through seeds, mechanical friction, fungi, insects, or soil and water sources.

[0545] The genome structures and replication mechanisms differ among genera within the family Tomato Cluster Dwarf Virus (TDRV). This discussion primarily focuses on the replication mode of the representative genus, *Tomato Cluster Dwarf Virus*. *Tomato Cluster Dwarf Virus* encodes two proteins involved in RNA replication: the 33K protein and the 92K protein. The former can also be referred to as the small replicase subunit SRS, and the latter is the RNA-dependent RNA polymerase RdRp. Similar to the genus *Tobacco Mosaic Virus*, RdRp enables viral genome replication in the protoplast, while both SRS and RdRp are indispensable in the plant. After the virus enters the plant cell, the viral mitochondria first disintegrate, releasing the viral genomic RNA. Subsequently, based on cap-independent translation, SRS and RdRp are expressed, and under the influence of a series of host factors, the viral positive RNA genome is replicated into antisense RNA. Finally, using the antisense RNA as a template, progeny viral RNA is synthesized. The progeny viral RNA assembles with the viral capsid protein to form viral mitochondria, which then carry out subsequent infection under the action of motility proteins.

[0546] In other exemplary embodiments, the viruses include those of the Benyviridae, Luteoviridae, and Solemoviridae families, which also utilize stop codon readthrough mechanisms to express their specific genes (e.g., extended capsid proteins), and the replication and spread of these viruses can be inhibited by antiviral tRNAs.

[0547] In some specific implementations, the virus includes at least one of Tobacco mosaic virus (TMV), Turnip vein-clearing virus (TVCV), Tobacco rattle virus (TRV), and Tomato bushy stunt virus (TBSV).

[0548] In some specific embodiments, the antiviral tRNA increases the RdRp / SRS translation ratio, thereby interfering with viral replication. In some specific embodiments, the antiviral tRNA inhibits the expression of viral coding genes. In some specific embodiments, the antiviral tRNA reduces viral titers. In some specific embodiments, the antiviral tRNA inhibits systemic viral infection.

[0549] In some embodiments, the present invention also provides an isolated polynucleotide, wherein the polynucleotide encodes the aforementioned antiviral tRNA.

[0550] In some embodiments, the present invention also provides a nucleic acid construct comprising the aforementioned polynucleotides.

[0551] In some embodiments, the present invention also provides a vector comprising the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0552] In some embodiments, the present invention also provides a recombinant host cell, wherein the recombinant host cell comprises the aforementioned antiviral tRNA, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned vector.

[0553] In some implementations, the host cell is derived from a plant.

[0554] In some embodiments, the present invention also provides the aforementioned antiviral tRNA, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned recombinant expression vector, or the aforementioned recombinant host cell for use in the following (a) and / or (b):

[0555] (a) Uses as an antiviral agent in plants;

[0556] (b) Use in the preparation of reagents for antiviral purposes in plants.

[0557] In some embodiments, the present invention also provides a method for improving plant resistance to viruses, the method comprising the steps of introducing the aforementioned antiviral tRNA, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned recombinant expression vector, or the aforementioned recombinant host cell into the plant.

[0558] The plants described in this invention can be monocotyledonous or dicotyledonous plants, as long as the aforementioned viruses can infect them. Suitable plants include, but are not limited to, corn, wheat, rice, barley, sorghum, beans, beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, and tobacco.

[0559] (NRC3 mutant)

[0560] In some implementations, the genetic element mutant includes a mutant of NRC3.

[0561] In some specific implementations, the wild-type NRC3 amino acid sequence is (SEQ ID NO:10; Nicotiana benthamiana):

[0562] In some embodiments, the present invention also provides a mutant of NRC3, wherein, compared with wild-type NRC3, the mutant reduces or eliminates the immunosuppression of SS15 secreted by pathogens in the plant.

[0563] In some specific embodiments, the amino acid sequence of SS15 is (SEQ ID NO:9; Globodera rostochiensis):

[0564] In some implementations, the mutant is selected from any one of the group consisting of (i)-(ii).

[0565] (i) A mutant comprising the sequence shown in SEQ ID NO:10, wherein the mutant has a mutated amino acid at one or more positions from position 308 to 383 of the sequence shown in SEQ ID NO:10;

[0566] (ii) The polypeptides shown in (i) have at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequences shown in (i).

[0567] In some preferred embodiments, the mutant has a mutated amino acid at one or more positions in the sequence shown in SEQ ID NO:10, from position 308 to 346.

[0568] In some preferred embodiments, the mutant corresponds to the sequence shown in SEQ ID NO:10 and has a mutated amino acid at any one position, or at any two or more positions, of the group consisting of (a1)-(a5):

[0569] (a1) The 312th amino acid, preferably K312P;

[0570] (a2) The 315th amino acid is preferably T315W;

[0571] (a3) The 316th amino acid is preferably E316R, E316K, or E316P;

[0572] (a4) The 317th amino acid is preferably N317K;

[0573] (a5) The 320th amino acid, preferably W320F.

[0574] In other embodiments, the mutant corresponding to the sequence shown in SEQ ID NO:10 also has a mutated amino acid at position (a6):

[0575] (a6) Amino acid at position 480, preferably D480V.

[0576] In some specific implementations, the mutant corresponds to the sequence shown in SEQ ID NO:10 and has the following characteristics: (m1)-(m 12Any of the mutated amino acids shown in )

[0577] (m1)E316R;

[0578] (m2)E316K;

[0579] (m3)T315W;

[0580] (m4)K312P;

[0581] (m5)E316P+W320F;

[0582] (m6)N317K+W320F;

[0583] (m7)E316R+D480V;

[0584] (m8)E316K+D480V;

[0585] (m9)T315W+D480V;

[0586] (m 10 K312P+D480V;

[0587] (m 11 E316P+W320F+D480V;

[0588] (m 12 )N317K+W320F+D480V.

[0589] In some embodiments, the present invention also provides an isolated polynucleotide, wherein the polynucleotide encodes a mutant of the aforementioned NRC3.

[0590] In some embodiments, the present invention also provides a nucleic acid construct comprising the aforementioned polynucleotides.

[0591] In some embodiments, the present invention also provides a vector comprising the aforementioned polynucleotide or the aforementioned nucleic acid construct.

[0592] In some embodiments, the present invention also provides a recombinant host cell, wherein the recombinant host cell comprises the aforementioned NRC3 mutant, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned vector.

[0593] In some implementations, the host cell is derived from a plant.

[0594] In some embodiments, the present invention also provides the use of the aforementioned NRC3 mutant, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned recombinant expression vector, or the aforementioned recombinant host cell in at least one of the following (a)-(d):

[0595] (a) Use in reducing or relieving immunosuppression of SS15 secreted by pathogens in plants;

[0596] (b) Use in the preparation of reagents for reducing or relieving immunosuppression of SS15 secreted by pathogens in plants;

[0597] (c) Uses in enhancing plant resistance to pathogens;

[0598] (d) Use in the preparation of reagents for enhancing plant resistance to pathogens.

[0599] In some implementations, the pathogens include secreted SS15 pathogens, such as the potato white nematode.

[0600] In some embodiments, the present invention also provides a method for improving plant resistance to pathogens, the method comprising the steps of introducing the aforementioned NRC3 mutant, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned recombinant expression vector, or the aforementioned recombinant host cell into the plant.

[0601] The plants described in this invention can be monocotyledonous or dicotyledonous plants, as long as the aforementioned pathogens can infect them. Suitable plants include, but are not limited to, corn, wheat, rice, barley, sorghum, kidney beans, sugar beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, and tobacco.

[0602] <Reagent Kit>

[0603] In some aspects, the present invention provides a kit comprising the controlled artificial replicon of geminiviruses as described in the <Controlled Artificial Replicon of Geminiviruses> section, the directed evolution / screening system of plants as described in the <Directed Evolution / Screening System in Plants> section, or a kit for carrying out the methods described in the present invention. Exemplarily, the kit may, for example, comprise a vector containing the controlled artificial replicon of geminiviruses, and / or a vector for expressing Rep and / or RepA proteins. The kit may also comprise instructions for carrying out the methods described in the present invention.

[0604] <Applications>

[0605] In some aspects of the invention, the use of the controlled artificial replicon of a geminivirus as described in the <Controlled Artificial Replicon of Geminiviruses> section, or the directed evolution / screening system of a plant as described in the <Directed Evolution / Screening System in Plants> section, is provided in obtaining mutants of the genetic element having the desired function.

[0606] In some aspects of the invention, the application of genetic element mutants or their coding sequences obtained by employing the plant-based directed evolution / screening system described in the "Plant-based Directed Evolution / Screening System" section of the invention or by employing the plant-based directed evolution / screening method described in the "Plant-based Directed Evolution / Screening Method" section of the invention in plants, particularly in plant genetic engineering.

[0607] Example

[0608] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0609] Materials and Methods

[0610] 1. Experimental materials

[0611] 1.1 Plant materials

[0612] The plant material used in the following examples is the Nicotiana benthamiana LAB strain. The seeds of Nicotiana benthamiana were preserved in our laboratory.

[0613] 1.2. E. coli strains

[0614] The Escherichia coli strains used in the following examples were DH5α, Fast-T1, and XL10. DH5α electrotransformed competent cells were purchased from TaKaRa (#Code No. 9027). Fast-T1 and XL10 chemically transformed competent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd. (Fast-T1, Vazyme, C505-02; XL10, Vazyme, C503-03).

[0615] 1.3 Agrobacterium strains

[0616] The Agrobacterium strains used in the following examples are EHA105 (Beijing Bomaide Gene Technology Co., Ltd., BC307-01), GV3101 (Beijing Bomaide Gene Technology Co., Ltd., BC308-01), and EHA105V (EHA105 containing the pSRK2 helper plasmid, constructed by the present invention).

[0617] 1.4 Experimental Carrier

[0618] The vectors used in the following examples are pCambia (provided and stored by our laboratory) and pPhi. Unless otherwise specified, the vector used in this document is pPhi.

[0619] 1.5 Commonly Used Molecular Biology Reagents

[0620] 2×Rapid Taq Master Mix for standard PCR was purchased from Novizan (P222-04). 2×Phanta Max Master Mix (Dye Plus) for high-fidelity PCR was purchased from Novizan (P525-03). Taq Pro Universal SYBR qPCR Master Mix for real-time PCR was purchased from Novizan (Q712-03). FastPure Plasmid Mini Kit for plasmid extraction was purchased from Novizan (DC201-01). ClonExpress Ultra One Step Cloning Kit was purchased from Novizan (C115-02). T4 DNA ligase was purchased from NEB (M0202L), and BsaI restriction enzyme was also purchased. DNA was purchased from NEB (R3733L), and 5′-adenosine triphosphate (ATP) was purchased from NEB (P0756S). A DNA agarose gel extraction kit was used by GeneJET. TM The Gel Extraction Kit was purchased from Thermo Scientific (K0692). The TaKaRa MiniBEST Plant RNA Extraction Kit was purchased from TaKaRa (#Code No. 9769). The RNA Reverse Transcription Kit... One-Step gDNA Removal and cDNA Synthesis SuperMix was purchased from TransGen Biotech (AT311-04). Dual-Luciferase Assay Kit The Reporter Assay System was purchased from Promega (E1980). Other relevant chemical reagents were either domestically produced analytical grade products or imported products.

[0621] 2. Experimental Methods

[0622] 2.1 Preparation of reagents and culture media

[0623] 2.1.1 Preparation of basic reagents and culture media

[0624] Preparation of LB liquid medium: Weigh 5g of yeast extract, 10g of tryptone, and 10g of sodium chloride (NaCl), dissolve in deionized water, and bring the volume to 1L. Autoclave at 121℃ for 20 minutes, then cool to room temperature before storage. Before use, add the appropriate working concentration of antibiotic and mix well. Store the LB liquid medium containing antibiotics at 4℃.

[0625] Preparation of LB solid medium: Weigh 5g yeast extract, 10g tryptone, 10g sodium chloride (NaCl), and 15g agar powder. Dissolve in deionized water and bring the volume to 1L. Autoclave at 121℃ for 20 minutes. Cool to ~60℃, add the appropriate working concentration of antibiotic, mix well, and pour into petri dishes, approximately 20ml per dish. After the solid medium has solidified, seal with sealing film and store upside down at 4℃.

[0626] Preparation of antibiotic stock solutions. Weigh the appropriate amounts of antibiotics, dissolve them in double-distilled water (DMSO for rifampin), and aliquot to the final volume. The stock solution concentrations for each antibiotic are as follows: Ampicillin: 100 mg / ml; Kanamycin: 10 mg / ml; Spectinomycin: 50 mg / ml; Gentamicin: 50 mg / ml; Rifampin: 50 mg / ml.

[0627] Preparation of 50×TAE electrophoresis buffer stock solution: Weigh or measure 242 g of tris(hydroxymethyl)aminomethane (Tris), 57.1 mL of glacial acetic acid, and 100 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH = 8.0). Stir to dissolve and mix thoroughly. Add deionized water and bring the volume to 1 L. Store at room temperature. When using, dilute the solution with deionized water to prepare 1×TAE buffer.

[0628] 2.1.2 Preparation of CTAB lysis buffer

[0629] CTAB lysis buffer is used for the extraction of plant DNA. Weigh 16.364 g sodium chloride, 20 ml 1 M Tris-HCl (pH = 8.0), 8 ml 0.5 M EDTA (pH = 8.0), and 40 g cetyltrimethylammonium bromide (CTAB). Add double-distilled water, dissolve, and bring the volume to 200 ml.

[0630] 2.1.3 Preparation of Tobacco Infection Solution

[0631] Tobacco infection solution was used for the rapid transformation of tobacco using the Agrobacterium tumefaciens infiltration method. Weigh 10g MgCl2·6H2O and 10g MES, dissolve them in double-distilled water, adjust the pH to 5.6-5.8 with NaOH, and bring the volume to 5L. Dispense the solution. The final concentration of the tobacco infection solution was 10mM MgCl2 and 10ml MES.

[0632] 2.1.4 Preparation of Acetyleugenone Mother Liquor

[0633] Acetosyringone was used to activate Agrobacterium. 0.392 g of acetosyringone was weighed, dissolved in anhydrous ethanol, and brought to a final volume of 10 ml. The concentration of the acetosyringone stock solution was 200 mg / ml.

[0634] 2.2 Plant Tissue DNA Extraction

[0635] The following examples demonstrate the extraction of plant tissue DNA using the CTAB method. The specific process is as follows:

[0636] 1. Collect the plant tissue in a 2ml grinding tube and add 1-2 stainless steel beads. Place the grinding tube in liquid nitrogen for flash freezing and grind using a SPEX SamplePrep 2010 Geno / Grinder grinder.

[0637] 2. After the plant tissue has returned to room temperature, add 800 μL of CTAB extraction solution to the grinding tube. Shake thoroughly to mix. Incubate in a 65°C oven for approximately 30 minutes.

[0638] 3. After the mixture has returned to room temperature, add 500 μL of chloroform to the grinding tube. Shake thoroughly to mix. The liquid will separate into layers at this point. Incubate at room temperature for approximately 10 minutes. Centrifuge at 12000 rpm for 5 minutes.

[0639] 4. After centrifugation, the liquid will separate into three layers: an upper aqueous phase, a middle layer of plant tissue residue and proteins, and a lower chloroform phase. Transfer approximately 700 μL of the upper aqueous phase to a new 1.5 ml centrifuge tube and add 500 μL of isopropanol. Invert the tube to mix thoroughly. DNA flocculent precipitate will appear at this point. Centrifuge at 12000 rpm for 5 minutes.

[0640] 5. Discard the supernatant. Add 500 μL of 75% ethanol. Shake well to mix. Centrifuge at 12000 rpm for 5 minutes.

[0641] 6. Repeat step 5.

[0642] 7. Discard the supernatant. Allow the remaining ethanol in the centrifuge tube to dry. Then add ~100 μL of double-distilled water. Store at 4°C or proceed with subsequent experiments.

[0643] 2.3 Total RNA extraction from plant tissues

[0644] The following examples demonstrate the extraction of total RNA from plant tissues using the TaKaRa MiniBEST Plant RNA Extraction Kit.

[0645] 2.4 Reverse transcription of RNA

[0646] The following examples utilize One-Step gDNA Removal and cDNA Synthesis SuperMix Kit for Reverse Transcription of Plant RNA.

[0647] 2.5 Carrier Construction

[0648] The following examples employ a reverse PCR strategy to construct the vector. The vector backbone and ligation fragments were amplified using 2×Phanta Max Master Mix high-fidelity PCR premix, and the ClonExpress Ultra One Step Cloning Kit and Golden Gate PCR kit were used. The Golden Gate Assembly Kit completes the construction of the carrier.

[0649] 2.6 Preparation of Agrobacterium electrotransformation competent cells

[0650] 1. Spread the Agrobacterium strain on a solid culture medium containing rifampin and the corresponding antibiotic.

[0651] 2.48 hours later, pick a single colony and incubate it overnight in a 50ml centrifuge tube (containing 10-15ml of liquid culture medium containing rifampicin and the corresponding antibiotic).

[0652] 3. Transfer the Agrobacterium tumefaciens bacterial suspension at a ratio of 1:100 to 1:300 to 300 ml of liquid culture medium containing rifampicin and the corresponding antibiotic, and incubate overnight.

[0653] 4. Once the Agrobacterium has been cultured to approximately OD = 1.0, transfer the bacterial culture to a 50ml centrifuge tube and centrifuge at 12000rpm for 5 minutes. Collect the bacterial cells and discard the culture medium.

[0654] 5. Add 10 ml of sterile double-distilled water to the bacterial cells and gently resuspend them. Then add more sterile double-distilled water to a final volume of 50 ml. Centrifuge at 12000 rpm for 5 minutes. Collect the bacterial cells and discard the liquid.

[0655] 6. Repeat step 5.

[0656] 7. Resuspend the bacterial cells in 20% sterile glycerol to a suitable volume.

[0657] 8. Aliquot the Agrobacterium bacterial culture. Quick-freeze in liquid nitrogen and store at -80°C.

[0658] 2.7. Agrobacterium electroconversion

[0659] 1. Remove the Agrobacterium electroconversion competent cells and thaw them on ice. Dilute the competent cells to the appropriate volume with sterile double-distilled water. Remove the electroconversion cup and drain the ethanol.

[0660] 2. Pipette 1 μL (~100 ng) of the plasmid to be transformed into a 1.5 ml centrifuge tube.

[0661] 3. Take 75 μL of Agrobacterium competent cells, mix them thoroughly with the plasmid, and add them to the electroporation cuvette.

[0662] 4. The electroconverter is set to E. coli electric shock mode.

[0663] 5. If the electrical pulse duration is longer than 2 ms, the electroconversion is successful (ideally, the electrical pulse duration should be longer than 4 ms). Aspirate the Agrobacterium tumefaciens culture. Add 500 ml of antibiotic-free LB liquid medium and incubate at 28°C for 1 hour.

[0664] 6. If transforming a single vector, take ~50 μL of the revived Agrobacterium culture and spread it on LB solid medium containing rifampicin and the appropriate antibiotic, and incubate at 28°C. If transforming a vector library, take ~10 μL of the revived Agrobacterium culture, spread it, calculate the colony-forming units, and add the remaining culture directly to 100 ml of LB liquid medium containing rifampicin and the appropriate antibiotic, and incubate at 28°C.

[0665] 2.8. Instantaneous transformation of tobacco using Agrobacterium infiltration method

[0666] 1. Inoculate Agrobacterium containing the target vector into LB liquid medium with the corresponding resistance and incubate overnight at 28°C.

[0667] 2. Add 200 μM acetylsuccine (1:1000 dilution of stock solution) to the tobacco staining solution.

[0668] 3. Centrifuge the Agrobacterium tumefaciens culture at 12,000 rpm for 5 minutes. Collect the bacterial cells and discard the culture medium. Add an equal volume of tobacco infection solution. Shake to resuspend.

[0669] 4. In a 2ml centrifuge tube, mix the individual Agrobacterium species according to the desired final OD value. Add tobacco infection solution to 1ml and mix well. Incubate at room temperature for ~30min.

[0670] 5. Select fully expanded tobacco leaves 5-6 weeks after sowing. Using a 1ml syringe (without the needle), inject Agrobacterium-methyl solution into the tobacco leaves. For general experiments, inject approximately 1cm... 2 For leaf tissue, the entire leaf needs to be injected for library screening.

[0671] 6. Two to four days after injection, observe the fluorescence as needed, or collect the leaves for subsequent experiments.

[0672] 2.9 Dual-luciferase assay

[0673] In the following examples, a dual-luciferase assay was used to assess the expression level of the target gene and the reading efficiency of the stop codon. The dual-luciferase assay was performed using equipment from Promega. The Reporter Assay System kit was completed. Luciferase activity was determined by... Measured with a 20 / 20 Luminometer.

[0674] 1. Add 10 ml of Luciferase Assay Buffer II to one bottle of Luciferase Assay Substrate. Add 0.2 ml of René Luciferase 50X Stop& Add 10ml of Stop& to Substrate Buffer.

[0675] 2. Four days after injecting tobacco, collect leaf tissue into a 2ml grinding tube and add 1-2 stainless steel beads. Grind the leaf tissue thoroughly with liquid nitrogen.

[0676] 3. After the cells have returned to room temperature, add 500 μL of 1X Passive Lysis Buffer (diluted from 5X Passive Lysis Buffer) to the grinding tube. Vortex to resuspend and mix, then lyse the cells.

[0677] 4. Transfer 20 μL of tobacco cell lysate to a 1.5 ml clear centrifuge tube, add 50 μL of firefly luciferase substrate, mix well, and measure the firefly luciferase activity.

[0678] 5. Add 50 μL of Renaissance luciferase substrate to the product from the previous step, mix well by whisking, and measure the Renaissance luciferase activity.

[0679] FLuc / RLuc is the ratio of the activity of firefly luciferase (FLuc) to that of kidney luciferase (RLuc) in the sample.

[0680] The stop codon readout efficiency is the ratio between the sample FLuc / RLuc value and the positive control FLuc / RLuc value.

[0681] 2.10 Real-time quantitative PCR

[0682] In the following examples, real-time quantitative PCR (qPCR) was used to determine the copy numbers of various vectors in bacteria, the copy numbers of geminivirus replicons in tobacco leaves, and the titers of RNA viruses. The real-time quantitative PCR experiments were performed using the premixed solution Taq Pro Universal SYBR qPCR Master Mix.

[0683] 1. Mix all components of the qPCR mixture. 2 μL diluted genomic DNA or cDNA, 2 μL forward primer, 2 μL reverse primer, 10 μL Taq Pro Universal SYBR qPCR Master Mix, and 4 μL double-distilled water. Add all components to a 96-blank white PCR plate.

[0684] 2. Set up the qPCR program. 95℃ for 60s (polymerase activation and DNA pre-denaturation), (95℃ for 5s, 60℃ for 15s) repeat for 41 cycles, and finally 65–95℃, increasing by 0.5℃ every 5s.

[0685] 3. Start the qPCR program and record the cycle number (Ct) at which the fluorescence threshold is reached.

[0686] 4. Calculate copy number and titer (2 -ΔCt (Method). Copy number, titer = 2^(Ct value of internal reference gene - Ct value of target sequence). For the copy number of geminivirus replicons, the internal reference gene is NbZDS1 (Nbv6.1trP36953); for the titer of RNA viruses, the internal reference gene is NbEF1α (Nbv6.1trP57121); for the copy number of plasmids in E. coli, the internal reference gene is the genome of E. coli; for the copy number of plasmids in Agrobacterium, the internal reference gene is the Ti plasmid of Agrobacterium coli.

[0687] 2.11. Fluorescent photography

[0688] Two to five days after Agrobacterium injection, the fluorescence of GFP and mSacrlet in tobacco leaves could be observed using a handheld UV lamp (LUYOR-3103P). The leaves could also be observed under a stereofluorescence microscope (OLYMPUS-SZX16). Image processing was performed using ImageJ 1.53e software.

[0689] 2.12. Diverse Barcode Designs

[0690] In the following examples, diversification barcodes (DB) are used to improve the diversity of the vector library. A diversification barcode is a 20 bp DNA segment containing discontinuous degenerate bases. To ensure no deviation occurs during PCR amplification, the GC content of the diversification barcode is controlled between 30% and 45%.

[0691] Example 1: Development of artificial replicons for twin viruses

[0692] 1. Development of self-replicating artificial replicons of twin viruses

[0693] To screen for the best-performing replicons in tobacco mesophyll cells, this embodiment first collected 19 geminiviruses from three genera: Begomovirus, Mastrevirus, and Curtovirus. These were then modified into self-replicating replicons, retaining most of the geminivirus elements while replacing the mobile protein (MP) and capsid protein (CP) (including the V3 gene for Curtovirus) – which are not directly related to replication – with a GFP expression cassette. Thus, these geminivirus replicons, containing replication proteins (Rep / RepA for Mastrevirus, and C1–C4 genes for Begomovirus and Curtovirus), can self-replicate and express GFP in plant cells.

[0694] In this embodiment, these 19 self-replicating artificial replicons were then constructed into the Agrobacterium vector pPhi. To ensure that the circular DNA replicons could be released from the linear Agrobacterium T-DNA, an extra copy of the viral LIR (or IR) was made, and the two LIR copies were loaded onto the flanking sides of the replicons. Thus, under the action of the Rep protein, the replicons could be released from the linear T-DNA and circularized (see Figure 1).

[0695] Next, in this embodiment, these self-replicating replicon vectors were transformed into Agrobacterium strain EHA105V and co-injected with the gene silencing inhibitor TBSVp19 (SEQ ID NO:6) into Nicotiana benthamiana. Four days later, DNA was extracted from plant mesophyll cells, and the copy number of the replicons was determined using qPCR.

[0696] As shown in Figure 2, most artificial replicons were able to undergo rolling circle replication in tobacco mesophyll cells. Among them, Bean Yellow Dwarf Virus (BeYDV), a virus from the genus Maize Stripe Virus, had the highest copy number, reaching approximately 40,000 copies per cell. Its nucleotide sequence is shown in SEQ ID NO:19. This experiment demonstrates that self-replicating geminivirus replicons can be generated in tobacco mesophyll cells via Agrobacterium infiltration and achieve efficient rolling circle replication, with BeYDV exhibiting the highest replication efficiency.

[0697] 2. Development of controlled artificial replicons for twin viruses

[0698] For directed evolution within plants, this self-replicating replicon is not optimal because the replication protein within it is expressed under viral element-driven conditions, thus making it impossible to control the level of rolling circle replication. Therefore, this embodiment attempts to remove the replication protein from the replicon. To this end, a series of vectors based on BeYDV were constructed and injected into *Nicotiana benthamiana*. DNA was extracted 4 days later, and the copy number was measured; the results are shown in Figure 3.

[0699] Experimental results showed that when Rep / RepA was absent (c1 in Figure 3; in subsequent examples, "Rep / RepA" refers to Rep and RepA, i.e., the presence of two proteins; unless otherwise specified, "Rep / RepA" also refers to the expression of Rep and RepA via alternative splicing in the expression cassette; the amino acid sequence of BeYDV Rep is shown in SEQ ID NO:3, the amino acid sequence of BeYDV RepA is shown in SEQ ID NO:4, and the nucleotide sequence of Rep and RepA expressed via alternative splicing is shown in SEQ ID NO:5), or when only RepA was present (c2 in Figure 3), the geminivirus replicon did not undergo rolling circle replication. However, when only Rep was present (c3 in Figure 3), the replicon underwent very limited rolling circle replication (approximately 200 copies per cell). This is because Rep cannot regulate the cell cycle of tobacco mesophyll cells, resulting in low expression levels of genes related to DNA replication in the cells, preventing the replicon from achieving efficient rolling circle replication. When Rep and RepA are present simultaneously, whether expressed separately via two expression cassettes (c4 in Figure 3) or via one expression cassette through alternative splicing (c5-c8 in Figure 3), the replicon undergoes highly efficient rolling circle replication, reaching approximately 50,000–80,000 copies per cell. Next, this example evaluated the role of the SIR in the replicon. Previous studies have shown that the SIR may be the binding site for endogenous primers during the conversion of geminiviruses from ssDNA to dsDNA. In this example, placing the SIR at various locations within the replicon revealed that the rolling circle replication levels of these replicons were similar. Subsequently, the SIR was removed, and rolling circle replication was not affected. This indicates that the SIR is not indispensable for rolling circle replication of the replicon. This is inconsistent with previous research, possibly because endogenous primers can also bind to other regions of the replicon, or the ssDNA to dsDNA conversion is not a critical process in these non-motile geminivirus replicons. Finally, in this embodiment, one LIR copy (c9 in Figure 3) was deleted. It was found that although the replication level of the replicon decreased significantly, it still remained at approximately 10,000 per cell. This may be because the entire pPhi vector was delivered into the plant cell by Agrobacterium and underwent rolling circle replication in the plant cell under the sole action of LIR. Based on the above results, this embodiment considers the eighth construction (c8 in Figure 3) to be the simplest at the same rolling circle replication level; therefore, subsequent modifications of the controlled replicons of other viruses were based on its construction method.

[0700] In the eighth construction, the structure of the controlled artificial replicon of the geminivirus is as follows:

[0701] [LIR]-[Target gene to be replicated]-[LIR].

[0702] As shown in Figure 4A, in this embodiment, 19 kinds of geminiviruses were modified into controlled replicons, that is, all viral sequences except LIR were deleted from the virus, and only one GFP expression frame was inserted (for example, the nucleotide sequence of BeYDV is shown in SEQ ID NO:20). The replication protein (Rep / RepA or C1~C4 protein) was driven by the Arabidopsis AtACT2 promoter in another vector.

[0703] As shown in Figure 4B, in this embodiment, these vectors were transformed into tobacco mesophyll cells, and it was found that when the replication protein was absent, none of the replicons underwent rolling circle replication; however, when the replicons were expressed, some viral replicons underwent significant rolling circle replication, with BeYDV still having the highest copy number, reaching approximately 90,000 copies per cell, an increase of approximately 1100 times compared to the form without the replication protein. However, some geminiviruses could perform relatively efficient rolling circle replication in the form of self-replicating replicons, but failed to do so in the form of controlled replicons. This may be because their replication protein was not properly expressed in the trans-expression state.

[0704] Based on these experimental results, this embodiment suggests that betaviruses can be modified into controlled replicons, whose rolling circle replication level can be regulated by corresponding replication proteins. Since BeYDV exhibits the highest copy number in both replicons and its replication proteins consist only of Rep and RepA, subsequent studies will be based on this virus.

[0705] 3. Establishment of the report replicon for rolling circle replication

[0706] Next, this embodiment aims to construct a reporter replicon to indicate rolling circle replication in plant cells. In the experiments described above, GFP was inserted into the replicon as a complete expression frame, meaning that GFP expression would occur even if rolling circle replication did not take place, although the green fluorescence would be stronger when rolling circle replication occurred. Therefore, this embodiment aims to establish a reporter replicon that only reports fluorescence when it undergoes rolling circle replication. To this end, this embodiment inserts an artificial intron into the GFP coding sequence, constructing the N-terminus of GFP and the 5' end of the artificial intron at the posterior end of the replicon, and the 3' end of the artificial intron and the C-terminus of GFP at the anterior end of the replicon (i.e., the seventh construction in Figure 3), as shown in Figure 5A. Thus, when rolling circle replication is absent, GFP cannot form a complete coding frame and therefore cannot report fluorescence; however, when rolling circle replication occurs, the two LIRs merge, and the presence of the artificial intron causes the LIR to be recognized as a single intron and spliced ​​after transcription. The N-terminus and C-terminus of GFP merge into a complete coding sequence, thus reporting fluorescence. In this embodiment, the reporter replicon is referred to as rearranged GFP (raGFP).

[0707] In this embodiment, the structure of the report replicon is as follows:

[0708] [LIR]-[3' end of the artificial intron]-[C-terminal coding sequence of the fluorescent protein]-[Terminator]-[SIR]-[Promoter]-[N-terminal coding sequence of the fluorescent protein]-[5' end of the artificial intron]-[LIR]

[0709] Its nucleotide sequence contains the nucleotide sequence shown in SEQ ID NO:18.

[0710] In this embodiment, the rolling circle replication reporter replicon was subsequently transformed into tobacco leaves. No fluorescence was produced in the absence of Rep / RepA; however, a very strong green fluorescence was observed when Rep / RepA was co-injected (see Figure 5, B). This is consistent with the expected results. The raGFP replicon was also used as a basal replicon in subsequent studies to investigate the level of rolling circle replication.

[0711] 4. The relationship between the size of twin virus replicons and their copy number

[0712] Finally, this embodiment aims to investigate the relationship between geminivirus replicons and their copy numbers. To this end, seven replicons of different sizes, ranging from 2.0 kb to 10.0 kb, were constructed and transformed into *Nicotiana benthamiana*. Four days after *Agrobacterium* injection, DNA was extracted and copy numbers were measured. The results showed that rolling circle replication did not occur in the absence of Rep / RepA; however, when Rep / RepA was added, the seven replicons exhibited different levels of rolling circle replication. The smallest 2.0 kb replicon reached approximately 60,000 copies per cell, while the largest 10.0 kb replicon only had approximately 6,000 copies (see Figure 6). This result indicates a negative correlation between the copy number and size of geminivirus replicons. Nevertheless, the 10 kb replicon replicated approximately 300-fold under the influence of Rep / RepA, a replication level sufficient for in vivo screening of the target gene.

[0713] This embodiment next evaluated the stability of replicons of different sizes. After high-throughput sequencing of the sample DNA, this embodiment calculated the coverage at each position by ligating the sequencing results with the replicon sequences, and further calculated the relative coverage. The results are shown in Figure 6. It can be seen that although the copy number of larger replicons is lower, their relative coverage can still be maintained above 75%, which indicates that the increase in size of geminivirus replicons does not seriously affect their replication stability in plant cells.

[0714] This experiment demonstrates that the in vivo directed evolution system assisted by twin viruses is not strictly limited by the size of the target gene.

[0715] Example 2: Establishment of a high-throughput screening system in plants

[0716] In addition to constructing artificial replicons of twin viruses, this embodiment also requires the establishment of a high-throughput screening system within plants to achieve directed evolution in plants.

[0717] The first step in this embodiment is to determine the upper limit of throughput achievable by the Agrobacterium tumefaciens introgression transformation method on tobacco leaves. To this end, this embodiment inserts a diversification barcode (DB) into a controlled replicon of a geminivirus (i.e., the eighth construction in "2. Development of a controlled artificial replicon of a geminivirus" in Example 1 (c8 in Figure 3)) to construct a replicon library. The diversification barcode consists of a series of discontinuous degenerate bases and has no biological function; its purpose is only to generate diversity to calculate throughput. Therefore, this library is a non-selective replicon library. Furthermore, the library size can be adjusted by changing the number of degenerate bases analyzed later; therefore, this library can be considered as a series of replicon libraries with different library sizes.

[0718] After the Golden Gate ligation was completed, the non-selective replicon library was electroporated into *E. coli*, followed by plasmid extraction and electroporation into *Agrobacterium*. Finally, the vector was co-injected into a leaf of *Nicotiana benthamiana* with constitutively expressed Rep / RepA driven by the NOS promoter at different *Agrobacterium* OD values. The leaf was collected 4 days later (see Figure 7). This embodiment sampled the Golden Gate ligation product, *E. coli*, *Agrobacterium*, and plant leaf replicons at four stages throughout the process, and performed deep sequencing on the samples. The proportion of each diverse barcode in each sample was then analyzed. By comparing the barcode proportion with that in the initial ligation product, this embodiment found that the overall distribution of all barcodes remained unchanged in the *E. coli* and *Agrobacterium* stages, reflecting a high R0. 2 The values ​​indicate that the library does not undergo perturbation during these two stages. However, during the plant leaf replicon stage (see Figure 8), when the OD of the injected Agrobacterium is high and the library size is small, there is a high linear correlation between the proportion of all barcodes and the initial ligation product; but when the OD of the injected Agrobacterium is low and the library size is large, this linear correlation disappears. This indicates that in this case, the library undergoes significant perturbation even without selection pressure, and the results of in vivo screening under such conditions are naturally less reliable. This is because the number of plant cells transformed by Agrobacterium is too small to meet the requirements of a library of this size.

[0719] Overall, as the OD of injected Agrobacterium decreased and the library size increased, the barcode distribution of the plant leaf replication stage gradually became disordered, reflected in R... 2 The value and coverage decrease. Referring to Figure 8, this embodiment can find a boundary where, under the conditions at its upper left, the sample's R... 2The high values ​​and coverage indicate that the library barcode distribution is the same as initially. This boundary also determines the minimum OD value for Agrobacterium injection when the library size is constant.

[0720] Next, this embodiment aims to confirm whether the tobacco leaf system can achieve in vivo screening with the assistance of geminivirus replicons. To this end, a screening library was constructed in this embodiment. The replication protein Rep / RepA was inserted into the replicons (i.e., the self-replicating artificial replicons from BeYDV constructed in the section "1. Development of Self-Replicating Artificial Replicons of Geminiviruses" in Example 1), and its motif I, a protein motif involved in DNA binding, was mutated. Based on prior results, these mutations were classified into three categories: functional mutations, non-functional mutations, and unclassified mutations. Theoretically, replicons containing the functional Rep / RepA mutation can achieve self-circular replication, thus enriching the entire library; while replicons containing the non-functional Rep / RepA mutation cannot achieve circular replication, resulting in depletion of the entire library. Meanwhile, this embodiment also inserts an intron into the encoding sequence of Rep / RepA, and inserts a diversified barcode into the intron to adjust the library size of the screening library, see Figure 9.

[0721] In this embodiment, the library was then subjected to the same procedures as the non-selective library, and samples were taken at four stages: Golden gate ligation product, *E. coli*, *Agrobacterium*, and plant leaf replicons. Deep sequencing was then performed on the samples to analyze the proportion of each variant-barcode. It can be seen that the library remained stable in both the *E. coli* and *Agrobacterium* stages, with R... 2 The values ​​remained at a very high level, indicating that no selection occurred. During the plant leaf replicon stage (see Figure 10), when the OD value was low, functional mutations were significantly enriched, while loss-of-function mutations were significantly depleted; however, when the OD value was high, no significant selection of mutations occurred, reflected in a higher R value. 2 This is because if the replicon containing the loss-of-function mutation enters a plant cell at the same time as the replicon containing the functional mutation, it can also undergo rolling circle replication, also known as the "hitchhiking effect".

[0722] Overall, the robustness of library screening decreased with increasing Agrobacterium injection OD values, but stabilized when OD < 0.03 (R0.03). 2<0.1), indicating that under these conditions, most tobacco mesophyll cells are transformed by only one Agrobacterium. Referring to Figure 10, this embodiment can also be roughly identified from Figure 10. Within this boundary, the screening robustness is very high, and the coverage can also be maintained at a high level, indicating that under these conditions, tobacco leaves can be used for in vivo screening of the target gene.

[0723] To further verify this conclusion, referring to Figure 11, this embodiment designed a dual-fluorescence experiment: the GFP expression frame in the BeYDV self-replicating replicon (i.e., the BeYDV self-replicating artificial replicon constructed in the section "1. Development of Geminid Self-Replicating Artificial Replicon" in Example 1) was replaced with the mScarlet expression frame. Agrobacterium was mixed in equal proportions with both (the BeYDV self-replicating replicon containing GFP and the BeYDV self-replicating replicon containing mScarlet) and injected into *Nicotiana benthamiana* at different OD values. The replicons containing GFP and mScarlet can self-replicate after being transformed into plant cells, thus avoiding gene expression differences caused by different OD values. Fluorescence was observed after 3 days. Referring to Figure 11, this embodiment found that when the OD values ​​were 0.001 and 0.003, only a small number of cells were transformed, and the red and green fluorescence did not overlap at all. When the OD values ​​were 0.01 and 0.03, most cells in the field of view were transformed, but only a small amount of red and green fluorescence overlapped. When the OD value is greater than or equal to 0.1, almost all mesophyll cells are transformed, and the red and green fluorescence almost completely overlap.

[0724] To objectively and quantitatively measure the transformation efficiency corresponding to different Agrobacterium OD values, this embodiment also designed a tobacco gene editing experiment, because the gene editing efficiency is directly related to the Agrobacterium transformation efficiency. In this embodiment, a single-stranded guide RNA (sgRNA; NbSGS-sgRNA; SEQ ID NO:21) targeting the tobacco endogenous gene (suppressor of gene silencing 3, NbSGS3, Nbv6.1trP59394) was constructed into a BeYDV self-replicating replicon (i.e., in the self-replicating artificial replicon from BeYDV constructed in the section "1. Development of Gemini self-replicating artificial replicon" in Example 1, the GFP expression cassette was replaced with the sgRNA expression cassette), and it was injected into tobacco leaves with different OD values ​​and 0.2 OD 35Sp-SpCas9 (amino acid sequence: SEQ ID NO:22; nucleotide sequence SEQ ID NO:23). The replicon containing the sgRNA can self-replicate after being transformed into plant cells, thus avoiding gene expression differences caused by different OD values. Four days later, deep sequencing was used to determine gene editing efficiency, and the relative gene editing efficiency was calculated. Referring to Figure 11, this example showed that when the OD value was greater than 0.1, the relative gene editing efficiency remained at a very high level (greater than 80%). When the OD values ​​were 0.03 and 0.01, the relative gene editing efficiency decreased significantly, but still remained at an acceptable level (~40%). When the OD value further decreased, the relative gene editing efficiency dropped below 10%, indicating that most tobacco mesophyll cells were not transformed by the sgRNA at this point.

[0725] Dual fluorescence experiments and gene editing experiments demonstrated that when the OD value of Agrobacterium was maintained at 0.01–0.03, most tobacco mesophyll cells could be transformed by only one Agrobacterium, and the transformation efficiency could be maintained at about 40% of the maximum transformation efficiency.

[0726] Based on the results of the previous experiments with non-selective and selective libraries, this embodiment suggests that, with the help of artificial replicons of geminiviruses, a single tobacco leaf can complete 10 [replications / replications]. 5 The in vivo screening of variants can be completed in just 4 days. This is far faster and more efficient than current plant directed evolution systems.

[0727] Example 3: Coupling of the target gene with rolling circle replication of the replicon

[0728] The next step, in order to establish a directed evolution system within plants using geminiviruses, is to couple the expected function of the target gene (Gene-of-interest, GOI) with the rolling circle replication of the geminivirus replicon. In this way, variants of the target gene with the expected function can activate (or inhibit) their own rolling circle replication, thereby changing their proportion in the library, while variants without the expected function will not. To this end, this implementation designed two coupling approaches.

[0729] The first approach is to forward couple the expected function of the target gene with the rolling circle replication of the replicon (A in Figure 12). Previous results have shown that rolling circle replication does not occur at all when Rep / RepA is absent; however, when co-injected with Rep / RepA, the replicon can undergo highly efficient rolling circle replication, with copy number increases of up to ~1100-fold. Therefore, by coupling the expected function of the target gene with the expression of Rep / RepA, its coupling with the rolling circle replication of the replicon can be achieved. To verify the feasibility and universality of this approach, the following experiments were designed in this embodiment. All vectors in this section used the pPhi vector as the vector backbone.

[0730] (1) First, this embodiment attempts to conjugate the intein with Rep / RepA. Referring to B in Figure 12, in order to conjugate the function of the intein with Rep / RepA, this embodiment divides the Rep / RepA protein into N-terminus and C-terminus at C183, and fuses them with different N-inteins (NpuDna-N-Intein, M86-N-Intein, NrdJ1-N-Intein, SspGyrB-N-Intein, sequences as shown in SEQ ID NO:32-35) and different C-inteins (NpuDna-C-Intein, M86-C-Intein, NrdJ1-C-Intein, SspGyrB-C-Intein, sequences as shown in SEQ ID NO:28-31). Functional and mutually recognizing N-inpeptides and C-inpeptides interact within plant cells, linking their exopeptides, Rep / RepA, into a complete protein. In this example, four inpeptides were separated and fused to the N- and C-termini of the Rep / RepA protein, and then co-injected into Nicotiana benthamiana with the reporter replicon raGFP (constructed in "3. Establishment of the reporter replicon for rolling circle replication" in Example 1). Four days later, replicon copy number measurements revealed that mutually recognizing inpeptides activated very strong rolling circle replication, while those that did not recognize each other did not activate it. This experiment demonstrates that Rep / RepA expression can be positively coupled to inpeptide function, and that there is good orthogonality between N- and C-inpeptides.

[0731] (2) Subsequently, this embodiment attempts to couple a sequence-specific protease to Rep / RepA. To couple the expression of Rep / RepA with the sequence-specific proteases (PVY-Nla-protease, TuMV-Nla-protease, TEV-Nla-protease, TVMV-Nla-protease, abbreviated as PVY, TuMV, TEV, and TVMV, respectively, with sequences shown in SEQ ID NO:36-39 and recognizing amino acid sequences shown in SEQ ID NO:40-43), this embodiment fuses a degrader (Degron; RDDK-degron; SEQ ID NO:44) to the N-terminus of Rep / RepA, and connects Rep / RepA to the degrader using a peptide recognizable by the specific protease (C in Figure 12). The presence of the degrader significantly reduces the half-life of the fused protein, causing it to be rapidly degraded by the host after translation. Since the Rep protein plays a crucial role in rolling circle replication, its half-life is naturally closely related to the level of rolling circle replication. In this example, a sequence-specific protease, the degrader-Rep / RepA, and the reporter replicon raGFP (constructed in "3. Establishment of the reporter replicon for rolling circle replication" in Example 1) were co-injected into *Nicotiana benthamiana*. Four days later, the replicon copy number was measured, revealing that the sequence-specific protease, in addition to recognizing and cleaving its corresponding peptide, could sometimes also recognize and cleave peptides that were not its corresponding peptide, thereby activating rolling circle replication (C in Figure 12). This indicates that the interaction between the two may not be specific enough. This experiment demonstrates that the expression of Rep / RepA can be positively coupled with the function of the sequence-specific protease, and that there is good orthogonality between some specific proteases and peptides.

[0732] (3) This embodiment attempts to couple a sequence-specific recombinase with Rep / RepA. To couple the expression of the sequence-specific recombinases (Cre, φC31, Flp, Bxb1; sequences shown in (SEQ ID NO: 24-26)) with Rep / RepA, this embodiment inserts two recombinase recognition sites at the 5' end of the Rep / RepA coding sequence, and inserts a terminator and a stop codon (D in Figure 12). Thus, when the recombinase is absent, both Rep / RepA transcription and translation terminate between the two recombinase recognition sites; when the recombinase is present and functional, recombination occurs at the two recombinase recognition sites, the terminator and stop codon are removed, Rep / RepA is expressed normally, and rolling circle replication is activated. In this embodiment, the recombinase, Rep / RepA construction, and the reporter replicon raGFP (constructed in "3. Establishment of the reporter replicon for rolling circle replication" in Example 1) are co-injected into *Tobacco Benzoenta*. Four days later, the copy number of the replicon was measured, revealing that the recombinase could complete recombination at its corresponding recognition site, activating strong rolling circle replication, but could not achieve recombination at other sites (D in Figure 12). This experiment demonstrates that the expression of Rep / RepA can be positively coupled with the function of the recombinase, and that there is a good orthogonality between the recombinase and its recognition site.

[0733] (4) This embodiment attempts to couple a self-cleavage peptide with Rep / RepA. Similar to the protease example, to couple the self-cleavage peptide with the expression of Rep / RepA, this embodiment fuses a degrader to the N-terminus of Rep / RepA and connects Rep / RepA to the degrader (RDDK-degron) using self-cleavage peptides (P2A, T2A, E2A, F2A, BmIFV2A, PVY-P1, PVY-HCpro; sequences shown in SEQ ID NO:47-53, respectively) (E in Figure 12; positive PC represents normally expressed Rep / RepA, while negative NC represents no added Rep / RepA). Thus, the active self-cleavage peptide can cleave the degrader, thereby stabilizing Rep / RepA and activating the rolling circle replication of the replicon. In this embodiment, the self-cleaving peptide-Rep / RepA was constructed and co-injected with the reporter replicon raGFP (constructed in "3. Establishment of the reporter replicon for rolling circle replication" in Example 1) into *Nicotiana benthamiana*. Four days later, the replicon copy number was measured, revealing that most of the self-cleaving peptides were functional, with E2A showing the highest efficiency. However, XTEN (XTEN linker; SEQ ID NO:46), used as a negative control, failed to self-cleave and activate rolling circle replication. This experiment demonstrates that the expression of Rep / RepA can be positively coupled to the function of the self-cleaving peptide.

[0734] (5) In this embodiment, we attempt to couple a transcriptional activator with Rep / RepA. To couple the transcriptional activator with the expression of Rep / RepA, we replace the Rep / RepA promoter with the Arabidopsis HTR10 promoter, which is hardly expressed in mesophyll cells, and insert a tandem repeat of six yeast GAL4 recognition sequences (UAS; SEQ ID NO:84) upstream of its core promoter. Then, we fuse the transcriptional activator with the DNA-binding domain (DNA motif; SEQ ID NO:80) of GAL4 (F to the left in Figure 12) to form GAL4-VPR (SEQ ID NO:81), GAL4-VP64 (SEQ ID NO:82), and GAL4-ERF2-AD (SEQ ID NO:83), respectively. Thus, GAL4 can recruit transcription activators upstream of Rep / RepA, and active transcription activators in plants can initiate Rep / RepA expression, thereby activating rolling circle replication. In this example, GAL4-transcription activator, Rep / RepA construction, and reporter replicon raGFP (constructed in "3. Establishment of reporter replicon for rolling circle replication" in Example 1) were co-injected into Nicotiana benthamiana. Four days later, replicon copy number measurements revealed that with only Rep / RepA construction and only GAL4 addition, replicon copy numbers were at very low levels. However, with the addition of GAL4-transcription activator, replicon copy number increased significantly, with the transcriptional activation domain (ERF2-AD) of Arabidopsis thaliana ethylene response factor 2 (ERF2-AD) showing the best effect (right side of F in Figure 12). This experiment demonstrates that Rep / RepA expression can be positively coupled with the function of transcription activators.

[0735] (6) Finally, based on the transcription activator, this embodiment attempts to couple a DNA binding protein with Rep / RepA. Similar to the transcription activator, in order to couple the expression of the DNA binding protein with Rep / RepA, this embodiment inserts tandem repeats of different DNA motifs (IcaRO, LmrAO, McbRO, PhlFO, and QacRO, sequences shown in SEQ ID NO:85-89, and GAL4-UAS) upstream of the core sequence of the Arabidopsis HTR10 promoter, and fuses the different DNA binding proteins with the transcription activation domain (ERF2-AD) of ethylene response factor 2 (left of F in Figure 12) to form IcaR-ERF2, LmrA-ERF2, McbR-ERF2, PhlF-ERF2, QacR-ERF2, sequences shown in SEQ ID NO:75-79, and GAL4-ERF2 (SEQ ID NO:74), respectively. Thus, DNA-binding proteins capable of recognizing specific motifs can activate Rep / RepA expression, thereby activating rolling circle replication. In this example, the DNA-binding protein-ERF2-AD fusion protein, Rep / RepA constructs containing different DNA motifs, and the reporter replicon raGFP (constructed in "3. Establishment of the reporter replicon for rolling circle replication" in Example 1) were co-injected into Nicotiana benthamiana. Four days later, the replicon copy number was measured, revealing that the DNA-binding proteins could recognize their corresponding DNA motifs and activate rolling circle replication (G in Figure 12). However, some DNA-binding proteins could also recognize non-corresponding DNA motifs, indicating a lack of strong orthogonality between them. This experiment demonstrates that Rep / RepA expression can be positively coupled to the function of DNA-binding proteins.

[0736] The above cases (1) to (6) demonstrate the feasibility of forward coupling of the expected function of the target gene with the rolling circle replication of the replicon. Furthermore, since Rep / RepA expression can be coupled with many different types of target genes, this method has good universality. These target genes can be used as candidate genes, and forward selection can be used to achieve directed evolution.

[0737] However, not all target genes can be coupled with Rep / RepA expression. Therefore, this embodiment proposes a second coupling approach: negatively coupling the expected function of the target gene with rolling circle replication of the replicon (Figure 13). Since rolling circle replication is a complex biological process involving many factors, target genes whose expected functions can directly or indirectly inhibit rolling circle replication can be coupled using this method. To verify the feasibility of this approach, the following experiments were designed in this embodiment.

[0738] (7) First, this embodiment attempts to couple a homologous recombination factor with rolling circle replication. In this embodiment, DNA exonucleases (mmExoI, trex2, t5 exo; sequences shown in SEQ ID NO:54-56, respectively), DNA helicase (RecQ; SEQ ID NO:57), DNA dissociation enzyme (RuvC; SEQ ID NO:57), single-stranded DNA annealing protein (SSAP; SEQ ID NO:59), single-stranded DNA binding protein (SSB; SEQ ID NO:60), DNA translocase (RecG; SEQ ID NO:61), and DNA recombinase (RecA; SEQ ID NO:62) were co-injected with constitutively expressed Rep / RepA and reporter replicon raGFP (constructed in "3. Establishment of reporter replicon for rolling circle replication" in Example 1) into *Tobacco Benedict's tobacco. Four days later, the copy number of replicons was measured, revealing that some DNA recombination factors significantly reduced the replicon copy number, especially the DNA annealing protein SSAP (Figure 13, B). This may be because these DNA recombination factors interfere with the DNA recombination process during rolling circle replication. These results demonstrate that the intended function of DNA recombination factors can be negatively coupled with rolling circle replication of the replicons.

[0739] (8) Next, this embodiment attempts to couple DNA deaminases with rolling circle replication. In this embodiment, DNA cytosine deaminases (A3A, eA3A, APOBEC1, A3B-ctd, PmCDA1, AIDx, miniSdd6, miniSdd7; sequences as shown in SEQ ID NO:63-70, respectively) and DNA adenine deaminase (ABE8e; SEQ ID NO:71) were co-injected into *Nicotiana benthamiana* with constitutively expressed Rep / RepA constructs and reporter replicons raGFP (constructed in "3. Establishment of reporter replicons for rolling circle replication" in Example 1). Four days later, the copy number of replicons was measured, and it was found that some DNA deaminases could significantly reduce the copy number level of replicons, especially cytosine deaminase A3A (C in Figure 13). The reason may be that these DNA deaminases use single-stranded DNA as a substrate for deamination and have mutations in the replication origin region (LIR) of the replicons, causing rolling circle replication to fail. The experimental results demonstrate that the expected function of DNA deaminase can be negatively coupled with rolling circle replication of the replicon.

[0740] (9) Finally, this embodiment attempts to couple the plant immunity elicitor with rolling circle replication. In this embodiment, the immune elicitors Xa10 (SEQ ID NO:72), Xa23 (SEQ ID NO:73), and Rx / CP were co-injected into Nicotiana benthamiana with constitutively expressed Rep / RepA and reporter replicon raGFP (constructed in "3. Establishment of reporter replicon for rolling circle replication" in Example 1). Four days later, the copy number of the replicon was measured, and it was found that all three could significantly reduce the copy number level of the replicon (D in Figure 13). The experimental results show that the expected function of the plant immunity elicitor can be negatively coupled with the rolling circle replication of the replicon.

[0741] These results demonstrate that the rolling circle replication of geminivirus replicons is a versatile platform that can be coupled to various types of target genes in both forward and reverse directions.

[0742] The results above demonstrate that geminitroviruses were developed into artificial replicons, that *Nicotiana benthamiana* can be used for high-throughput in vivo plant screening and that its operational parameters were determined, and that the biological functions of various target genes can be coupled with rolling circle replication of the replicons. Based on these findings, a directed evolutionary system for plants assisted by geminitrovirus replicons (GRAPE) was conceived (Figure 14). In GRAPE, the target gene, after in vitro mutagenesis, is inserted into a geminitrovirus replicon to construct a mutant library of the target gene. Then, based on the expected function of the target gene, a genetic pathway is designed to couple the target gene with rolling circle replication of the replicons. After transforming the mutant library and other elements into *Nicotiana benthamiana* using Agrobacterium tumefaciens infiltration, the tobacco leaves are collected and DNA is extracted after 3-4 days of reaction. High-throughput sequencing and calculation of the changes in each variant within the library identify the target gene variants that meet the requirements. Because GRAPE works within plant systems, it is well-suited for the directed evolution of plant-specific genes.

[0743] Example 4: Evolution of anti-viral tRNA using GRAPE

[0744] 1. The ratio between SRS and RdRp is crucial for viral replication.

[0745] Among plant viruses, those from the Virgaviridae and Tombusviridae families cause severe crop diseases and yield losses every year. Developing resistance genes for these two types of viruses is of great significance in crop breeding. Viruses from these two families share a common structural feature: they both require two protein components to replicate their RNA genomes: RNA-dependent RNA polymerase (RdRp) and a small replicase subunit (SRS). SRS and RdRp share the N-terminus, and RdRp is expressed through a leaky stop codon in the genome. Previous studies have shown that the expression ratio between SRS and RdRp is crucial for viral replication.

[0746] To confirm this, this embodiment first inserted a GFP into the genomes of four Virgaviridae viruses: Tobacco mosaic virus (TMV), Turnip vein-clearing virus (TVCV), Tobacco rattle virus (TRV), and Tomato bushy stunt virus (TBSV). Referring to Figure 15, for TMV-GFP, this embodiment replaced the coding sequence of its capsid protein with GFP. To ensure its mobility, this embodiment added the capsid protein and 3'UTR sequence of Tobacco mild green mosaic virus (TMGMV) to the 3' end of the virus. For TVCV-GFP, this embodiment replaced the coding sequence of its capsid protein with GFP. Similarly, in this embodiment, the capsid protein and 3'UTR sequence of TGMV are added to the 3' end of the virus, and a precise viral 3' end is generated using a hammerhead ribozyme of Tobacco ringspot virus (TRSV). For TRV-GFP, the coding sequence of the TRV2 2c protein is replaced with GFP. For TBSV-GFP, a portion of the coding sequence of the TBSV capsid protein is replaced with GFP, and a precise viral 3' end is generated using a Hepatitis delta virus ribozyme (HRV-Rz). These recombinant viral infectious clones can infect Tobacco Bengal and express the GFP protein.

[0747] To confirm that the expression ratio between SRS and RdRp is crucial for viral replication, this embodiment replaced the leaky stop codons in the TVCV-GFP and TBSV-GFP constructions with TAA and TGG, respectively. The TAA stop codon reduces readthrough, while the TGG tryptophan codon ensures complete readthrough. Injecting these viral constructions into *Nicotiana benthamiana* revealed that the TAA stop codon significantly reduced TVCV-GFP replication, but had little effect on TBSV, possibly because the low RdRp expression level under TAA conditions was sufficient for viral replication. However, the TGG tryptophan codon strongly inhibited replication in both viruses (see Figure 16). This experimental result is consistent with previous reports. Therefore, this embodiment concludes that efficient readthrough of leaky stop codons in viral RdRp can inhibit viral replication.

[0748] Based on the above results, this embodiment hypothesizes whether a suppressor tRNA (sup-tRNA) can be used to promote the reading of stop codons, thereby enabling plants to develop resistance to viruses (Figure 17). Suppressor tRNAs are a type of tRNA that recognizes stop codons through base complementarity and inhibits translation termination when the ribosome encounters a stop codon during translation. Suppressor tRNAs are encoded in the plant nuclear genome, chloroplast genome, and mitochondrial genome, and can correct missense or nonsense mutations to a certain extent, ensuring correct protein synthesis. In this embodiment, this tRNA that can promote stop codon reading and inhibit viral replication is called anti-viral tRNA.

[0749] 2. Testing for repressive tRNAs in plants and animals

[0750] This embodiment first constructs a dual-luciferase-based readthrough reporter system, Termination-FLuc (A in Figure 18). In this embodiment, a stop codon and a P2A self-cleaving peptide are inserted at the N-terminus of the FLuc coding sequence. To avoid potential ribosomal leakage scanning-induced FLuc leakage expression, a tandem initiation codon (TIC; SEQ ID NO:46) is inserted before the stop codon. Thus, FLuc can only be expressed when the stop codon is read through.

[0751] This embodiment first tested artificially repressive tRNAs used in mammalian tRNA therapy. However, only very low readthrough efficiency was observed, with the highest being only about 10%. This may indicate that repressive tRNAs in animals are not compatible with plant systems. Next, this embodiment tested reported endogenous repressive tRNAs in plants. However, no FLuc expression was observed in any of these embodiments, demonstrating that overexpression of endogenous repressive tRNAs does not produce stop codon readthrough (see B and C in Figure 18).

[0752] However, this result contradicts reality. Under natural conditions, viruses require endogenous repressive tRNA to read the stop codon and express RdRp. The fact that the virus can replicate normally in tobacco indicates that the endogenous repressive tRNA is active. To explain this contradiction, this embodiment added viral sequences to both sides of the stop codon in termination-FLuc to simulate the viral sequence environment. This embodiment then transformed the relevant vector into *Tobacco Bengal* using the Agrobacterium infiltration method. The results showed that FLuc expression could be detected in the TMV sequence environment (Figure 19). This indicates that the endogenous repressive tRNA is active, but requires a specific sequence environment and its activity is low.

[0753] tRNA undergoes complex post-transcriptional processing and modification during expression, and interacts with endogenous ribosomes, amino acid-tRNA aminoacyltransferases, and ribosomal RNA. Therefore, this embodiment plans to use the GRAPE system to evolve anti-viral tRNA with high readability suitable for plant systems.

[0754] Therefore, this embodiment first needs to couple the anti-viral tRNA with the rolling circle replication of the biviral replicon. To this end, this embodiment uses the pPhi vector as a backbone, inserting a stop codon at the N-terminus of the Rep / RepA coding sequence, mimicking termination-FLuc, to construct termination-Rep (A in Figure 20; sequence as shown in SEQ ID NO: 90-92). This embodiment tested termination-Rep with repressive tRNA from mammals, finding that termination-Rep did not induce rolling circle replication when transformed into tobacco with wild-type tRNA, but rolling circle replication occurred when stop codon readthrough occurred. The copy number level matched the stop codon readthrough efficiency (B in Figure 20). Adding viral sequences to the flanking sides of the stop codon in termination-Rep also showed the same trend as termination-FLuc (sequence as shown in SEQ ID NO: 93-95) under the action of endogenous repressive tRNA (Figure 20). Therefore, this embodiment suggests that termination-Rep can couple the function of anti-viral tRNA with replicon rolling circle replication.

[0755] Subsequently, this embodiment collected 32 high-copy-rate tRNAs from Solanaceae plants from the PlantRNA 2.0 database, changed their anticodons to CUA, UCA, UUA, and CCA, introduced a small number of mutations into their backbones, and inserted them into a geminivirus artificial replicon (i.e., the eighth construction in "2. Development of Geminivirus Controlled Artificial Replicon" in Example 1 (c8 in Figure 3)). Using the pPhi vector as the backbone, the first anti-viral tRNA library was constructed using the Golden Gate ligation method (Figure 21). In this library, only anti-viral tRNAs that can induce stop codon readthrough can activate rolling circle replication, thereby enriching themselves in the library (Figure 21).

[0756] In this embodiment, the library was co-injected with *Nicotiana benthamiana* along with Termination-Rep vectors of three different stop codon types. Four days later, tobacco DNA was extracted, deep sequenced, and the proportion of each variant in the initial library and replicons was compared. As shown in Figure 22, some anti-viral tRNAs were functional, but most were inactive.

[0757] To obtain anti-viral tRNAs with higher readability, this embodiment further mutated the backbones of five tRNAs that performed well in the previous evolutionary round, constructing a second anti-viral tRNA library (Figure 22). This library was then co-injected into *Nicotiana benthamiana* with three different termination codon types of Termination-Rep, followed by deep sequencing four days later. The results showed that when co-injected with Termination-Rep in the TAG and TGA termination codon forms, a large number of anti-viral tRNAs with anticodons CUA and UCA, respectively, were enriched in the library. This suggests the effectiveness of the evolution (Figure 22). However, when the TAA form of Termination-Rep was used, almost no anti-viral tRNAs were enriched.

[0758] To verify the results of directed evolution, this embodiment selected a series of highly enriched anti-viral tRNAs (SEQ ID NO: 98-207), constructed vectors for each, and measured their stop codon reading activity using Termination-FLuc (Figure 23). The results showed that the anti-viral tRNAs with CUA and UCA anticodons indeed induced very efficient stop codon reading, reaching up to ~90%. However, the anti-viral tRNA with UUA anticodons only induced ~10% reading efficiency. This is consistent with the results of directed evolution. The predicted secondary structures of some evolved anti-viral tRNAs are shown in Figure 24.

[0759] This embodiment aims to investigate whether these evolved anti-viral tRNAs can alter the expression ratio of viral SRS / RdRp. Since the translation efficiency of SRS and RdRp is difficult to measure directly, this embodiment expresses RLuc (SEQ ID NO:8) and FLuc (SEQ ID NO:7) in tandem, adding a viral stop codon and its surrounding 48 bp. This embodiment uses this construct to simulate the expression of viral SRS and RdRp, using the luciferase activity ratio of FLuc / RLuc to represent the translation ratio of RdRp / SRS. A construct with the stop codon replaced by TGG was used as a positive control (without added tRNA). This embodiment co-injects this construct with anti-viral tRNA into *Nicotiana benthamiana*, and the luciferase activities of FLuc and RLuc were measured 4 days later. The results showed that the anti-viral tRNA significantly increased FLuc / RLuc, suggesting that it could also increase the translation ratio of RdRp / SRS, thereby interfering with viral replication (see Figure 25).

[0760] Next, to confirm the antiviral activity of anti-viral tRNA, in this embodiment, anti-viral tRNA was co-injected with TMV-GFP, TVCV-GFP, TBSV-GFP, and TRV-GFP into *Nicotiana benthamiana*. For TMV-GFP and TVCV-GFP, the anti-viral tRNA was injected simultaneously with the viral infectious clone; for TBSV-GFP and TRV-GFP, the anti-viral tRNA was injected one day prior to the viral infectious clone. The results showed that, compared to native tRNA, anti-viral tRNA significantly reduced the fluorescence level of GFP, indicating that anti-viral tRNA can inhibit the expression of viral coding genes (see Figure 26).

[0761] To further confirm that anti-viral tRNA can inhibit viral replication, RNA was extracted from the injection site tissue in this embodiment, and the viral titer was detected using RT-qPCR. This embodiment utilized multiple pairs of qPCR primers to detect the titer of a single virus, comprehensively reflecting the levels of RNA in its various subgenomic regions. The results showed that, compared to native tRNA, anti-viral tRNA significantly reduced the viral titer, particularly for TBSV (see Figure 27).

[0762] Next, this embodiment aims to test whether anti-viral tRNA can inhibit systemic viral infection. In this embodiment, natural tRNA or anti-viral tRNA was first injected into tobacco leaves, and one day later, an infectious TMV-GFP clone was injected at the same location. This embodiment found that under the natural tRNA condition, TMV systemic infection occurred 5 days after injection. Seven days after injection, all seven injected tobacco plants were systemically infected. However, under the anti-viral tRNA condition, only one plant showed systemic infection 13 days after injection, while the remaining plants remained uninfected. This result indicates that anti-viral tRNA can significantly inhibit systemic viral infection (see Figure 28).

[0763] Finally, this embodiment aims to further confirm that anti-viral tRNA inhibits viral replication by promoting the readthrough of the stop codon in the viral RdRp. To this end, an anti-viral tRNA-virus orthogonal experiment was designed. In Figure 29, this embodiment co-injected anti-viral tRNA with CUA anticodons with TMV, TVCV, and TBSV containing UAG stop codons in RdRp; and co-injected anti-viral tRNA with UCA anticodons with TRV containing UGA stop codons in RdRp into tobacco, achieving inhibition of viral replication. To demonstrate the working principle of anti-viral tRNA, this embodiment then co-injected anti-viral tRNA with CUA anticodons with TRV, and anti-viral tRNA with UCA anticodons with TMV, TVCV, and TBSV. The results showed that the anti-viral tRNA did not significantly reduce GFP fluorescence in either case, indicating that viral replication was not inhibited.

[0764] In this embodiment, anti-viral tRNA was co-injected with geminivirus BSCTV-GFP and non-viral form 35Sp-GFP to transform Nicotiana benthamiana. The results also showed that anti-viral tRNA did not significantly reduce GFP fluorescence, indicating that anti-viral tRNA does not interfere with the replication of geminivirus and does not affect the expression of ordinary genes (see Figure 29).

[0765] The above results demonstrate that anti-viral tRNA does indeed inhibit viral replication by prompting the readthrough of the stop codon in RdRp.

[0766] In this embodiment, the function of anti-viral tRNA was coupled with rolling circle replication of geminivirus replicons, and a series of anti-viral tRNAs that could efficiently promote stop codon readout were obtained through directed evolution using the GRAPE system. Subsequently, this embodiment demonstrated that anti-viral tRNAs can inhibit viral replication by promoting stop codon readout in the viral RdRp, thereby conferring broad-spectrum viral resistance to plants. This embodiment reveals for the first time that tRNA can be used to resist viral infection and provides a new strategy for the future development of virus-resistant crop varieties.

[0767] Compared to other antiviral strategies, antiviral tRNAs offer several advantages. First, a few antiviral tRNAs can confer broad-spectrum viral resistance in plants. Most members of the Virgaviridae and Tombusviridae families rely on stop codon readthrough to express SRS and RdRp, which are crucial for their replication. Theoretically, antiviral tRNAs could inhibit the replication of all these viruses. Furthermore, members of the Benyviridae, Luteoviridae, and Solemoviridae families also utilize stop codon readthrough mechanisms to express their specific genes (mostly extended capsid proteins), and the replication and spread of these viruses could also be inhibited by antiviral tRNAs. In contrast, traditional methods such as RNA silencing and the development of resistance receptors can only generate resistance against a few viruses; for new viruses, resistance genes need to be developed anew.

[0768] Secondly, the viral resistance established by anti-viral tRNA is durable. Viruses often mutate during replication, making it easy for them to escape recognition by host resistance proteins by introducing several mutations into their encoded proteins, thus reinfecting plants. However, anti-viral tRNA targets the viral gene expression mechanism, making it difficult for viruses to alter their gene expression strategies and thus difficult to escape recognition by anti-viral tRNA.

[0769] Third, anti-viral tRNA is very small, typically only about 80 nt, which makes it very easy to deliver into plant cells. Therefore, in addition to being stably transformed into the plant genome, anti-viral tRNA can also be delivered into plant cells through in vitro spraying or by means of nanoparticles.

[0770] Therefore, anti-viral tRNA provides a new strategy for future antiviral breeding in agriculture.

[0771] Example 5: Obtaining NRC3 mutants not suppressed by SS15 using GRAPE directed evolution.

[0772] 1. SS15 inhibits NRC3-mediated cell death.

[0773] Based on previous research, the effector protein SS15, derived from the potato white nematode (Globodera pallida), directly targets and inhibits the NRC3-dependent immune response in Nicotiana benthamiana (Derevnina et al., 2021) (A in Figure 30). In this embodiment, NRC3 is used as the screening target, and the goal is to use the GRAPE system to screen for NRC3 mutants that can relieve SS15 inhibition.

[0774] 1.1 SS15 inhibits NRC2 H480R and NRC3 D480V Instead of NRC4 D478V Induced cell death

[0775] Consistent with previous reports, single amino acid mutants such as NRC2 generated from the MHD motif of Nicotiana benthamiana NRC H480R NRC3 D480V and NRC4 D478V All of them can produce significant cell death, and compared to NRC3 D480V and NRC4 D478V Cell death caused by NRC2 H480R The induced cell death was relatively weak; furthermore, SS15 could significantly inhibit NRC2. H480R and NRC3 D480V Induced cell death, but did not inhibit NRC4 D478V This leads to cell death (B in Figure 30).

[0776] 2. SS15 inhibits Pto / AVRPto-NRC3 and Gpa2 / RBP1-NRC3-induced cell death.

[0777] This example demonstrates that SS15 suppresses the self-activated form of NRC3. D480V Cell death induced by NRC3 is induced by upstream-sensing NLRs, and some upstream-sensing NLR-induced cell death is dependent on NRC3. To further investigate whether upstream-sensing NLR-induced and NRC3-dependent cell death is inhibited by SS15, this study selected two downstream-sensing NLRs, NbPrf and Gpa2, and co-expressed them with NRC3 in the presence of their corresponding recognized pathogen effector proteins (or Pto complexes). Results after 3 days of tobacco injection showed that, compared to the control group, co-expression of NRC3 with Pto / AVRPto or Gpa2 / RBP1 induced significant cell death phenotypes. When SS15 was co-expressed, cell death induced by Pto / AVRPto and Gpa2 / RBP1 was significantly inhibited compared to the control group (Figure 31). This indicates that SS15 inhibits NRC3-dependent cell death induced by sensing NLRs.

[0778] 3. SS15 may interact with the HD1-1 domain of NRC3.

[0779] 3.1, NRC4 3HD1-1 It will be suppressed by SS15

[0780] SS15 vs NRC3 D480V and NRC4 D478V The induced cell death exhibited inhibitory selectivity (B in Figure 30). To identify NRC3 domains sensitive to SS15 inhibition, this embodiment involved swapping different NRC3 domains with NRC4, and identifying SS15-sensitive NRC3 regions by observing whether co-expression of SS15 resulted in cell death inhibition (A in Figure 32). The results showed that, consistent with previous findings, SS15 inhibited NRC3... D480V Caused cell death, however NRC4 D478V Cell death induced by NRC4 was not inhibited by SS15 (B in Figure 30 and B in Figure 32). Transient expression results in tobacco after NRC3-NRC4 domain exchange showed that all six chimeric proteins generated by NRC4 exchange of NRC3 domains resulted in loss of cell death induction (V1-V6 and V8). Among them, a new NRC chimeric protein, NRC4, was found to be generated by replacing the first 39 amino acids (a308-a346, named 3HD1-1) of the NRC3 HD domain (a307-a381) with the first 39 amino acids (a307-a345, named 4HD1-1) of the NRC4 HD domain (a307-a381). 3HD1-1 (V7) caused significant cell death in tobacco cells, and NRC4 3HD1-1 Induced cell death was inhibited by SS15 (B in Figure 32). This indicates that NRC4 3HD1-1 The recognition of SS15 means that the HD1-1 region of NRC3 may interact with SS15.

[0781] 3.2 NRC3-mediated cell death reduces replicon copy number

[0782] To further verify NRC3 D480V Pto / AVRPto-NRC3-mediated cell death can reduce replicon copy number. In this example, the raGFP replicon (constructed in "3. Establishment of reporter replicon for rolling circle replication" in Example 1) was selected as the target and co-expressed with a series of expression vectors in wild-type Nicotiana benthamiana. Results 3 days after tobacco injection showed that, compared to the control group, NRC3... D480VPto / AVRPto-NRC3 induced significant cell death, but co-expression of SS15 significantly inhibited cell death and restored rolling circle replication of raGFP, producing green fluorescence (Figure 33, A). Cell death index statistics showed that NRC3... D480V The resulting cell death was more intense than that mediated by Pto / AVRPto-NRC3, and co-expression of SS15 significantly reduced the cell death index of both (Figure 33, B). raGFP replicon copy number measurements showed no significant difference in replicon copy number between tobacco expressing NRC3 and SS15 alone and the control group. Co-expression of Pto / AVRPto-NRC3 and NRC3 in tobacco... D480V This resulted in a 40.31-fold and 131.91-fold decrease in replicon copy number compared to the control group, respectively. When both were co-expressed with SS15, the replicon copy number recovered to a level comparable to the control group (C in Figure 33). This indicates that NRC D480V Both Pto / AVRPto-NRC3-induced cell death significantly reduced replicon copy number, and the degree of replicon reduction was significantly positively correlated with the degree of induced cell death.

[0783] 4. Obtain NRC3 mutants that are not suppressed by SS15 using GRAPE-directed evolution.

[0784] To identify NRC3 mutants that have been relieved of SS15 suppression, this embodiment attempts to use the established GRAPE system for negative directed evolution. NRC3 D480V The cell death induced by this method was significantly stronger than that induced by the sensed NLR (A and B in Figure 33), and considering the number of screening elements, NRC3 was selected in this embodiment. D480V It serves as an evolutionary chassis to obtain NRC3 mutants that are not suppressed by SS15.

[0785] 4.1, using NRC3 D480V Building a mutant library for the chassis

[0786] This embodiment demonstrates that SS15 suppresses the self-activated form of NRC3. D480V Induced cell death (B in Figure 30) and revealed that the HD1-1 region of NRC3 is involved in the response to SS15 (Figure 32). This example targets NRC3. D480V110 mutants were artificially designed in the HD region, including 82 single-amino acid mutations and 28 double-amino acid mutations, covering amino acids 304-348 of NRC3 (with the HD1-1 region located at amino acids 308-346). Each mutant was assigned 4 bars, and the wild-type was assigned 40 bars, resulting in a total of 480 bars. To avoid the added bars affecting protein function, introns were added to the coding sequence of NRC3 in this embodiment, and the barcode sequences were constructed within the intron sequences. For example, the nucleotide sequence of the NRC3 library is shown in SEQ ID NO:96 (Figure 34).

[0787] 4.2 Results of NRC3 Directed Evolution

[0788] Referring to Figure 35, further analysis of the high-throughput sequencing data shows that wild-type NRC3 D480V The proportion of barcodes corresponding to most NRC3 variants remained unchanged, while the proportion of some variants decreased. In addition, among the screened mutants, the E316P+N317K double mutant had the highest combined attenuation score, followed by the E316R and E316K single mutants.

[0789] 4.3, with NRC3 D480V Mutants screened for chassis validation

[0790] To ensure the robustness of the screening results, this embodiment verifies NRC3. D480V The mutants were selected negatively in the presence of SS15 (SEQ ID NO:9) as the chassis. Based on the pooled decay score (MDS) ranking, 10 mutants were constructed separately in this embodiment, combined with the control (NRC3). D480V The mutant, abbreviated as DV, also known as autoNRC3 or NbNRC3-D480V, has the sequence SEQ ID NO:10. It differs from the wild-type NRC3 (NbNRC3) shown in SEQ ID NO:10 by a mutation at position 480 (D mutated to V). It was co-expressed with SS15 in tobacco. Results 3 days after tobacco injection showed that, in addition to the three mutants previously confirmed to relieve SS15 inhibition (DV / E316P+N317K, DV / E316P, and DV / N317K), the remaining six mutants DV / E316R, DV / E316K, DV / T315W, DV / K312P, DV / E316P+W320F, and DV / N317K+W320F all relieved SS15 inhibition of NRC3. D480VThe inhibition of induced cell death was observed, and the mutant DV / L339D+P340E, with the lowest attenuation score among the 10 mutants, did not relieve the inhibition of SS15 (Figure 36). This result further demonstrates that the replicon-assisted screening system GRAPE can achieve negative screening of mutants of the target gene, and successfully screened 9 mutants that relieved the inhibition of NRC3 by SS15. D480V Mutants that induce plant immunity.

[0791] 4.4. Validation of mutants screened using NRC3 as a chassis

[0792] To further verify the inhibitory effect of the screened mutants on NRC3-dependent sensing NLR-induced cell death and to compare the de-inhibition effect on different sensing NLRs, this embodiment selected three sensing NLRs for experiments, including NRC2 / NRC3-dependent NbPrf and Gpa2, and NRC2 / NRC3 / NRC4-dependent Rx. Considering that SS15 inhibits the function of NRC2 / NRC3 but not NRC4 (B in Figure 30), this embodiment selected NbPrf (Pto / AVRPto, sequences as shown in SEQ ID NO:12 and 13, respectively) and Gpa2 / RBP1 (sequences as shown in SEQ ID NO:14 and 15, respectively) to verify the NRC3 mutant in wild-type tobacco, and selected Rx / CP (sequences as shown in SEQ ID NO:16 and 17, respectively) to verify the NRC3 mutant in nrc2 / 3 / 4 knockout tobacco.

[0793] 4.4.1. Expression of the NRC3 mutant alone does not induce cell death.

[0794] To investigate whether site mutations derived from wild-type NRC3 induce cell death, this study generated nine positive site mutations (Figure 36) from wild-type NRC3 (SEQ ID NO:10), excluding D480V. Specifically, these mutations were: E316P+N317K, E316P, N317K, E316R, E316K, T315W, K312P, E316P+W320F, and N317K+W320F. These mutations were then transiently expressed in tobacco to investigate whether they induced cell death. The results showed that 3 days after tobacco injection, compared to the positive control D480V (NRC3), cell death was significantly reduced. D480V The nine mutants tested did not induce a significant cell death phenotype when expressed transiently alone in tobacco (Figure 37).

[0795] 4.4.2 The NRC3 mutant relieved the inhibition of Pto / AVRPto-induced cell death by SS15.

[0796] In this embodiment, the interaction protein of the tobacco endogenous sensing NLR NbPrf was first selected to verify whether the selected mutation site could relieve the inhibition of SS15 in Pto / AVRPto-induced cell death. Three mutants (E316P+N317K, E316K, and T315W) were generated in wild-type NRC3 and co-expressed with Pto / AVRPto and SS15 in wild-type tobacco. The results after 3 days of tobacco injection showed that, compared with the wild-type NRC3 control group, all three NRC3 mutants significantly relieved the inhibition of Pto / AVRPto-induced cell death by SS15 (Figure 38).

[0797] 4.4.3 The NRC3 mutant relieves the inhibition of Gpa2 / RBP1-induced cell death by SS15.

[0798] Potato-derived sensitive NLR Gpa2 recognizes RBP1 secreted by *G. pallida* and activates downstream NRC2 or NRC3-mediated immune responses (Figure 38). In this example, three mutants (E316P+N317K, E316K, and T315W) verified in Figure 36 were generated in NRC3 and transiently co-expressed with Gpa2 / RBP1 and SS15 in wild-type tobacco. The results 3 days after tobacco injection showed that, consistent with the Pto / AVRPto verification results, all three mutants significantly relieved the inhibition of Pto / AVRPto-induced cell death by SS15 compared to the wild type (Figure 38).

[0799] 4.4.4 The NRC3 mutant relieves the inhibition of Rx / CP-induced cell death by SS15.

[0800] Rx recognizes viral-derived CP and induces selection-dependent cell death in three helper NLRs (NRC2 / NRC3 / NRC4). Similarly, in this example, three mutants (E316P+N317K, E316K, and T315W) validated in Figure 34 were generated in NRC3 and co-expressed with Rx / CP and SS15 in nrc2 / 3 / 4 knockout tobacco, respectively. The results after 3 days of tobacco injection showed that, compared with the wild-type NRC3 control group, all three mutants significantly relieved the inhibition of Rx / CP-induced cell death by SS15 (Figure 38).

[0801] 4.4.5. The screened NRC3 mutants did not relieve the functional inhibition of AVRcap1b.

[0802] Previous studies have found that the effector protein AVRcap1b, derived from Oomycete, inhibits NRC3-mediated immune responses through a membrane transport-associated protein, NbTOL9a. This mechanism differs from the functional inhibition produced by SS15 binding to NRC3 (Dong et al., 2021). To demonstrate that NRC3 mutants specifically relieve the inhibition of SS15-induced cell death, this study selected AVRcap1b as another inhibitory factor to generate three mutant forms, E316P+N317K, E316K, and T315W, as verified in Figure 36, in wild-type NRC3. These mutants were then co-expressed with Pto / AVRPto or Gpa2 / RBP1 in wild-type NRC3. Furthermore, the three NRC3 mutants were co-expressed with AVRcap1b and Rx / CP in nrc2 / 3 / 4 knockout tobacco, respectively. Results 3 days after tobacco injection showed that AVRcap1b significantly inhibited cell death induced by the three sensed NLRs (Figure 39, A), and none of the three NRC3 mutants significantly relieved the inhibition of cell death induced by AVRcap1b (Figure 39, A and B). This indicates that the screened NRC3 mutants specifically relieved the inhibition of cell death induced by SS15.

[0803] 5. The NRC3 mutants screened were deactivated from interacting with SS15.

[0804] To demonstrate the interaction between SS15 and NRC3, and to show that the screened NRC3 mutants eliminated the direct interaction with SS15, this study fused the transcriptional activation domain (AD) of GAL4 with SS15 and the DNA binding domain (BD) with different NRC3 mutants, including E316P+N317K, T315W, and W320F, for in vitro yeast interaction experiments. The yeast interaction results showed a significant interaction between SS15 and wild-type NRC3. The E316P+N317K double mutant and the T315W mutant significantly eliminated the interaction with SS15 (Figure 40), indicating a correlation between the SS15-NRC3 interaction and the SS15-NRC3 functional inhibition. Eliminating the SS15-NRC3 interaction can break the SS15-NRC3 functional inhibition.

[0805] To further demonstrate the interaction between NRC3 and SS15 in tobacco, this embodiment fused NRC3 and three mutants, E316P+N317K, T315W, and W320F, with the DNA-binding domain of yeast GAL4, respectively. SS15 was fused with the transcriptional activation domain VP64 for expression. After GAL4 and VP64 bind, the expression of the downstream reporter gene LUC or RUBY is transcribed and activated. LUC acts as a luciferase to catalyze the production of luciferin, while RUBY catalyzes the production of a visible metabolite, betaine. Three days after tobacco injection, fluorescein assays showed that, compared to the control group, co-expression of NRC3-GAL4 and W320F-GAL4 with SS15-VP64 both reported fluorescein, while co-expression of E316P+N317K-GAL4 and T315W-GAL4 showed no significant difference compared to the negative control (A and B in Figure 41). Ruby's report showed that, five days after tobacco injection, co-expression of NRC-GAL4 and W320F-GAL4 with SS15-VP64 both showed significant redness. The red phenotype was observed, while co-expression of E316P+N317K-GAL4 and T315W-GAL4 did not show a significant red phenotype (C in Figure 41). Spectrophotometric quantification of betaine showed that, compared to the control group, the absorbance of leaves co-expressed with NRC3-GAL4 and W320F-GAL4 was significantly higher than that of the negative control. The absorbance values ​​produced by co-expression of E316P+N317K-GAL4 and T315W-GAL4 with SS15-VP64 were not significantly different from those of the negative control (D in Figure 41). These results indicate a significant interaction between NRC3 and SS15 in *Tobacco Bengal*. The two mutant forms of NRC3, E316P+N317K and T315W, significantly dissolve the interaction with SS15, while the W320F mutation has no significant effect on the interaction between NRC3 and SS15.

[0806] 6. The screened NRC3 mutant restored the plant's resistance to *Pseudomonas syringae*.

[0807] Pseudomonas syringae secretes effector proteins AVRPto or AVRPtoB, which mediate NRC2- or NRC3-dependent immune responses after recognition by the Pto / Prf complex (Wu et al., 2021). The mutants screened in this embodiment can relieve the inhibition of Pto / AVRPto-NRC3-induced cell death by SS15. To further investigate whether the NRC3 mutant confers resistance to AVRPto-derived Pseudomonas syringae, this embodiment constructed an RFP-tagged DC3000 as the test pathogen. Its secreted effector protein AVRPto activates NbPrf (forming a complex with Pto) to mediate NRC3-dependent immunity. In this embodiment, Pto, SS15, and NRC3 mutants were transiently expressed in tobacco beforehand, and DC3000 was inoculated 3 days after tobacco injection. Three days after DC3000 inoculation, tobacco tissue of the same size inoculated pathogen area was quantitatively removed, ground, and the grinding solution was serially diluted and spotted in KB medium containing resistance. After pathogen growth culture for 2 days, the statistical results showed that there was no significant difference in the number of colonies among the five treatments in the control group (Figure 42, A). When SS15 was co-expressed, the number of colonies per unit area in the NRC3 and W320F treatment groups was higher. Compared with NRC3, the number of colonies per unit area in the E316P+N317K, E316K, and T315W mutant treatment groups was significantly reduced (Figure 42, B). This indicates that the NRC3 mutants E316P+N317K, E316K, and T315W relieved the cell death inhibition caused by SS15, significantly limited the growth of DC3000, and thus enhanced the resistance to the pathogen.

[0808] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0809] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0810] References

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[0816] Dong X,Derevnina L,Contreras MP,Adachi H,Upson J,Vergara Cruces A,Xie R, J,Menke FLH,Mugford ST,et al.Plant pathogens convergently evolved to counteract redundant nodes of an NLR immune receptor network.PLOS Biology.2021:19(8)

[0817] Gil-Humanes J,Wang Y,Liang Z,Shan Q,Ozuna CV,Sanchez-Leon S,Baltes NJ,Starker C,Barro F,Gao C,et al.High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR / Cas9.Plant J.2017:89(6):1251-1262.

[0818] Hager KJ,Perez Marc G,Gobeil P,Diaz RS,Heizer G,Llapur C,Makarkov AI,Vasconcellos E,Pillet S,Riera F,et al.Efficacy and Safety of a Recombinant Plant-Based Adjuvanted Covid-19 Vaccine.N Engl J Med.2022:386(22):2084-2096.

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[0821] Li C,Zhang R,Meng X,Chen S,Zong Y,Lu C,Qiu JL,Chen YH,Li J,and Gao C.Targeted,randommutagenesis of plant genes with dual cytosine and adenine base editors.Nat Biotechnol.2020:38(7):875-882.

[0822] Lozano-Duran R.Geminiviruses for biotechnology:the art of parasite taming.New Phytol.2016:210(1):58-64.

[0823] Mor TS,Moon YS,Palmer KE,and Mason HS.Geminivirus vectors for high-level expression of foreign proteins in plant cells.Biotechnol Bioeng.2003:81(4):430-437.

[0824] Ren Q,Sretenovic S,Liu S,Tang X,Huang L,He Y,Liu L,Guo Y,Zhong Z,Liu G,et al.PAM-less plant genome editing using a CRISPR-SpRY toolbox.Nat Plants.2021:7(1):25-33.

[0825] Rodriguez-Negrete E,Lozano-Duran R,Piedra-Aguilera A,Cruzado L,Bejarano ER,and Castillo AG.Geminivirus Rep protein interferes with the plant DNA methylation machinery and suppresses transcriptional gene silencing.New Phytol.2013:199(2):464-475.

[0826] Wang M,Lu Y,Botella JR,Mao Y,Hua K,and Zhu JK.Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR / Cas9 system.Mol Plant.2017:10(7):1007-1010.

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[0828] Yu Y, Wang

[0829] The sequence involved in this invention:

[0830] The following is the tRNA sequence.

Claims

1. A controlled artificial replicon of a geminivirus comprising two long intergenic regions (LIRs) derived from common soybean yellow dwarf virus (BeYDV), with the two LIRs located on either side of the artificial replicon. Optionally, the target gene to be replicated is contained between the two LIRs; Optionally, the nucleotide sequence of the LIR is selected from at least one of the following groups (i)-(iv): (i) Contains a nucleotide sequence as shown in SEQ ID NO:1; (ii) A mutant sequence of the nucleotide sequence shown in SEQ ID NO:1, wherein the mutant sequence has a mutated nucleotide at one or more positions corresponding to the sequence shown in SEQ ID NO:1, and the mutant sequence has the function or activity of the nucleotide sequence shown in SEQ ID NO:

1. (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii) and has the function or activity of the nucleotide sequence shown in SEQ ID NO:1; (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in SEQ ID NO:

1.

2. The geminivirus-controlled artificial replicon of claim 1, wherein, A short intergenic region (SIR) is included between the two LIRs; or, no SIR is included between the two LIRs.

3. The geminivirus-controlled artificial replicon according to claim 1 or 2, wherein, The target gene is operatively linked to the expression regulatory sequence.

4. The geminivirus-controlled artificial replicon according to any one of claims 1 to 3, wherein, The replication of the artificial replicon is controlled by Rep and / or RepA proteins derived from common soybean dwarf virus (BeYDV); Optionally, the replication of the artificial replicon is controlled by the Rep protein derived from common bean yellow dwarf virus (BeYDV); Preferably, the replication of the artificial replicon is controlled by the Rep and RepA proteins derived from common bean yellow dwarf virus (BeYDV).

5. The geminivirus-controlled artificial replicon of claim 4, wherein, The amino acid sequence of the Rep protein is selected from at least one of the following groups (i)-(iv): (i) Contains an amino acid sequence as shown in SEQ ID NO:3; (ii) An amino acid sequence having at least 70%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:3; (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:3, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:

3. (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:3 under stringent conditions, and said amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:3, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent. And / or, the amino acid sequence of the RepA protein is selected from at least one of the following groups (i)-(iv): (i) Contains an amino acid sequence as shown in SEQ ID NO:4; (ii) An amino acid sequence having at least 70%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:4, and retaining the function or activity of the amino acid sequence shown in SEQ ID NO:4; (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:4, and retains the function or activity of the amino acid sequence shown in SEQ ID NO:

4. (iv) An amino acid sequence encoded by a nucleotide sequence, said nucleotide sequence hybridizing with a polynucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO:4 under stringent conditions, and said amino acid sequence retaining the function or activity of the amino acid sequence as shown in SEQ ID NO:4, said stringent conditions being moderately stringent, moderately-highly stringent, highly stringent, or very highly stringent.

6. A plant-based directed evolution / screening system for directed evolution or screening of genetic elements to obtain mutants of said genetic element having a desired function, said plant-based directed evolution / screening system comprising: (i) a controlled artificial replicon of a twin virus as described in any one of claims 1 to 5; (ii) the Rep and / or RepA proteins as described in claim 4 or 5; and (iii) Mutants or mutant libraries of the genetic element; wherein The replication of the controlled artificial replicon of the twin virus is configured to be associated with the desired function of a mutant of the genetic element.

7. The in-plant directed evolution / screening system of claim 6, wherein, The genetic elements include protein-coding sequences, functional RNA-coding sequences, or expression-regulating sequences; and / or, The genetic elements are derived from plants or are intended to be applied to plants.

8. The in-plant directed evolution / screening system of claim 6 or 7, wherein, The replication of the controlled artificial replicon of the geminivirus includes the replication of the controlled artificial replicon of the geminivirus in plant cells; and / or, The plant-based directed evolution / screening system is used within plant cells; Optionally, the plant includes monocotyledonous plants or dicotyledonous plants; Optionally, the plants include corn, wheat, rice, barley, sorghum, beans, beets, tomatoes, cassava, cucumbers, Arabidopsis thaliana, or tobacco; Optionally, the plant cell is an isolated plant cell, a cell in a plant tissue, a cell in a plant organ, or a cell in a plant body.

9. The in-plant directed evolution / screening system according to any one of claims 6-8, wherein, The controlled artificial replicon of the geminivirus, the coding sequences of the Rep and / or RepA proteins, and the coding sequences of mutants of genetic elements or mutants in a mutant library are constructed into the vector.

10. The in-plant directed evolution / screening system according to any one of claims 6-9, wherein, The target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant from a mutant library; Optionally, the mutant library of the genetic element is obtained by inserting multiple mutants of the genetic element into a controlled artificial replicon of a geminivirus.

11. The in-plant directed evolution / screening system according to any one of claims 6 to 10, wherein, The replication of the controlled artificial replicon of the geminivirus is configured to be associated with the desired function of a mutant of the genetic element, including: setting the replication level of the controlled artificial replicon of the geminivirus to be associated with the desired function of a mutant of the genetic element, or setting the activity or expression level of Rep and / or RepA proteins in plant cells to be associated with the desired function of a mutant of the genetic element.

12. The in-plant directed evolution / screening system according to any one of claims 6-11, wherein, The plant-based directed evolution / screening system is used for directed evolution of antiviral tRNAs, including: (i) a vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant from a mutant library, wherein the genetic element is an antiviral tRNA to be evolved; and, (ii) A vector for expressing Rep and / or RepA proteins, wherein the expression cassette of Rep and / or RepA proteins contains: a tandem start codon (TIC), a stop codon, a self-cleaving peptide coding sequence, and a Rep and / or RepA protein coding sequence.

13. The in-plant directed evolution / screening system according to any one of claims 6-11, wherein, The plant-based directed evolution / screening system is used for the directed evolution of mutants of immune-related proteins in plants, wherein the mutants of immune-related proteins relieve the inhibition of proteins derived from pathogens, including: (i) A vector containing a controlled artificial replicon of a geminivirus, wherein the target gene to be replicated in the controlled artificial replicon of the geminivirus includes a mutant of the genetic element or a mutant in a mutant library, wherein the genetic element is the coding sequence of a mutant of the immune-related protein to be evolved; (ii) a vector expressing Rep and / or RepA proteins; and, (iii) A vector expressing the pathogen-derived protein, which has an inhibitory effect on innate / initiated immune-related proteins; Optionally, the immune-related protein includes proteins in the NLR network; preferably, the immune-related protein includes NRC. More preferably, the immune-related protein includes NRC3, and the pathogen-derived protein includes SS15.

14. The in-plant directed evolution / screening system of claim 13, wherein, The coding sequence of the NRC3 mutant to be evolved to remove SS15 inhibition contains an intron sequence, preferably, the intron sequence contains a DNA barcode.

15. The in-plant directed evolution / screening system according to any one of claims 6-11, wherein, The plant-based directed evolution / screening system is selected from any one of the following (A) to (F): (A) Plant in vivo directed evolution / screening systems for screening peptides, including: (i) A vector expressing a first fusion protein, the first fusion protein comprising the N-terminus of the inteptide to be screened, and the N-terminus or C-terminus of Rep and / or RepA protein; (ii) a vector expressing a second fusion protein, the second fusion protein comprising the C-terminus of the inteptide to be screened, and the C-terminus or N-terminus of Rep and / or RepA protein; and, (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. Wherein, the N-terminus or C-terminus of the Rep and / or RepA protein in the vector expressing the first fusion protein and the C-terminus or N-terminus of the Rep and / or RepA protein in the vector expressing the second fusion protein together form a complete Rep and / or RepA protein; (B) Plant-based directed evolution / screening systems for screening sequence-specific proteases, including: (i) A vector expressing the sequence-specific protease to be screened; (ii) a vector expressing a fusion protein, said fusion protein comprising, from N-terminus to C-terminus: a degrader, a peptide recognized by the sequence-specific protease to be screened, and Rep and / or RepA protein; and, (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (C) Plant-based directed evolution / screening systems for screening sequence-specific recombinases, including: (i) A vector expressing Rep and / or RepA proteins, the vector comprising a coding sequence of Rep and / or RepA proteins, and two recombinase recognition sites located at the 5' end of the coding sequence of Rep and / or RepA proteins, with a terminator and a stop codon between the two recombinase recognition sites; (ii) A vector expressing the sequence-specific recombinase to be screened, and, (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (D) Plant-based directed evolution / screening systems for screening self-cleaving peptides, including: (i) a vector for expressing a fusion protein, wherein the fusion protein comprises, from the N-terminus to the C-terminus: a degrader, a self-cleaving peptide to be screened, and Rep and / or RepA protein; and, (ii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (E) Plant-based directed evolution / screening systems for screening transcription activators, including: (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by a known DNA-binding domain, a recognition sequence of the transcription activator to be screened, and a sequence encoding Rep and / or RepA proteins. (ii) a vector expressing a fusion protein, said fusion protein comprising the selected transcription activator and the known DNA-binding domain, and, (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (F) Plant-based directed evolution / screening systems for screening DNA-binding proteins, including: (i) A vector expressing Rep and / or RepA proteins, including a DNA sequence or motif recognized by the DNA-binding domain to be screened, a recognition sequence of a known transcription activator, and a sequence encoding Rep and / or RepA proteins. (ii) a vector for expressing a fusion protein, said fusion protein comprising the known transcription activator and the DNA-binding domain to be screened, and, (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

16. The in-plant directed evolution / screening system according to any one of claims 6-11, wherein, The plant-based directed evolution / screening system is selected from any one of the following (G) to (I): (G) Plant-based directed evolution systems for screening DNA recombination factors, including: (i) Vectors expressing DNA recombinant factors to be evolved; (ii) A vector constitutively expressing Rep and / or RepA proteins; (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (H) Plant-based directed evolution systems for screening DNA deaminases, including: (i) A vector expressing DNA deaminases to be evolved; (ii) A vector constitutively expressing Rep and / or RepA proteins; (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode. (I) A plant-based directed evolution system for screening plant immune elicitors, including: (i) Vectors expressing plant immune elicitors to be evolved; (ii) A vector constitutively expressing Rep and / or RepA proteins; (iii) A vector containing a controlled artificial replicon of a geminivirus, wherein the replication level of the target gene to be replicated in the controlled artificial replicon of the geminivirus can be detected, and optionally, the target gene to be replicated includes a fluorescent protein coding sequence or a DNA barcode.

17. A method for directed evolution / screening in plants, wherein the method employs a directed evolution / screening system for plants as described in any one of claims 6 to 16 to perform directed evolution or screening of genetic elements to obtain mutants of the genetic elements having the desired function.

18. The plant in vivo directed evolution / screening method according to claim 17, comprising: The steps of introducing the plant in vivo directed evolution / screening system into a population of plant cells; and, The steps for culturing the population of plant cells; Preferably, the plant-based directed evolution / screening system is introduced into the plant cell population via Agrobacterium infiltration.

19. The plant in vivo directed evolution / screening method according to claim 17 or 18, further comprising: The steps include detecting and selecting genetic element mutants with the desired function in a population of said plant cells, and optionally, identifying the function of the selected genetic element mutants.

20. A genetic element mutant, obtained using the plant-based directed evolution / screening system as described in any one of claims 6 to 16, or by the plant-based directed evolution / screening system method as described in any one of claims 17 to 19.

21. The genetic element mutant according to claim 20, wherein the antiviral tRNA has a nucleotide sequence selected from at least one of the following groups (i)-(iv): (i) Contains a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153; (ii) A mutant sequence of a nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, wherein the mutant sequence has a mutated nucleotide at one or more positions of the sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153, and the mutant sequence has the function or activity of the nucleotide sequence as shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153; (iii) Under high-strict hybridization conditions or very high-strict hybridization conditions, it is able to be reverse complementary to the hybridization sequence of the nucleotide sequence shown in (i) or (ii), and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152 and 153. (iv) has at least 70%, optionally at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153, and has the function or activity of the nucleotide sequence shown in any one of SEQ ID NO: 106, 107, 138, 140, 141, 144, 148, 152, and 153.

22. The genetic element mutant of claim 21, wherein the virus is a virus whose replication and / or transmission are affected by stop codon readout; Optionally, the virus includes at least one of the following families: Virgaviridae, Tombusviridae, Benyviridae, Luteoviridae, and Solemoviridae. Preferably, the virus includes at least one virus from the family Virgaviridae and the family Tobusviridae; More preferably, the virus includes at least one of Tobacco mosaic virus (TMV), Turnip vein-clearing virus (TVCV), Tobacco rattle virus (TRV), and Tomato bushy stunt virus (TBSV).

23. A biological material selected from any one of (a) to (d): (a) an isolated polynucleotide, wherein, The polynucleotide encodes the antiviral tRNA as described in claim 21 or 22; (b) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide described in (a); (c) A vector, wherein the vector comprises the polynucleotide described in (a) or the nucleic acid construct described in (b); (d) Recombinant host cell, wherein the recombinant host cell comprises the antiviral tRNA of claim 21 or 22, the polynucleotide of claim (a), the nucleic acid construct of claim (b), or the vector of claim (c); preferably, the host cell is derived from a plant.

24. The use of the antiviral tRNA as described in claim 21 or 22, or the use of the biological material as described in claim 23, for antiviral purposes in plants; and / or for use in the preparation of reagents for antiviral purposes in plants.

25. A method for enhancing plant resistance to viruses, the method comprising the step of introducing antiviral tRNA as described in claim 21 or 22, and / or biological material as described in claim 23 into the plant.

26. The genetic element mutant of claim 20, wherein the mutant is an NRC3 mutant, which, compared with wild-type NRC3, reduces or relieves the immunosuppression of SS15 secreted by pathogens in plants; Optionally, the mutant is selected from any one of the following groups (i)-(ii); (i) A mutant comprising the sequence shown in SEQ ID NO:10, wherein the mutant has a mutated amino acid at one or more positions from position 308 to 383 of the sequence shown in SEQ ID NO:10; (ii) A polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (i); Preferably, the mutant has a mutated amino acid at one or more positions in the sequence shown in SEQ ID NO:10, from position 308 to 346; More preferably, the mutant corresponds to the sequence shown in SEQ ID NO:10 and has a mutated amino acid at any one position, or at any two or more positions, of the group consisting of (a1)-(a5): (a1) The 312th amino acid, preferably K312P; (a2) The 315th amino acid is preferably T315W; (a3) The 316th amino acid is preferably E316R, E316K, or E316P; (a4) The 317th amino acid is preferably N317K; (a5) The 320th amino acid, preferably W320F.

27. The genetic element mutant of claim 26, wherein, The mutant corresponds to the sequence shown in SEQ ID NO:10 and also has a mutated amino acid at position (a6): (a6) Amino acid at position 480, preferably D480V.

28. The genetic element mutant of claim 27, wherein, The mutant corresponds to the sequence shown in SEQ ID NO: 10, with the mutation in any one of the following (m1) - (m 12 ) shown in the amino acid: (m1)E316R; (m2)E316K; (m3)T315W; (m4)K312P; (m5)E316P+W320F; (m6)N317K+W320F; (m7)E316R+D480V; (m8)E316K+D480V; (m9)T315W+D480V; (m 10 ) K312P+D480V; (m 11 ) E316P + W320F + D480V; (m 12 ) N317K + W320F + D480V.

29. A biological material selected from any one of (a) to (d): (a) an isolated polynucleotide, wherein, The polynucleotide encodes a mutant of NRC3 as described in any one of claims 26 to 28; (b) A nucleic acid construct, wherein the nucleic acid construct comprises the polynucleotide described in (a); (c) A vector, wherein the vector comprises the polynucleotide described in (a) or the nucleic acid construct described in (b); (d) Recombinant host cell, wherein the recombinant host cell comprises a mutant of NRC3 as described in any one of claims 26 to 28, a polynucleotide as described in (a), a nucleic acid construct as described in (b), or a vector as described in (c); preferably, the host cell is derived from a plant.

30. The use of a mutant of NRC3 as described in any one of claims 26 to 28, or the use of the biological material as described in claim 29 in at least one of the following (a)-(d): (a) Use in reducing or relieving immunosuppression of SS15 secreted by pathogens in plants; (b) Use in the preparation of reagents for reducing or relieving immunosuppression of SS15 secreted by pathogens in plants; (c) Uses in enhancing plant resistance to pathogens; (d) Use in the preparation of reagents for enhancing plant resistance to pathogens; Preferably, the pathogen includes secreted SS15 pathogens, such as potato white nematode.

31. A method for improving plant resistance to pathogens, the method comprising the steps of introducing a mutant of NRC3 as described in any one of claims 26 to 28, and / or the biological material as described in claim 29 into the plant.

32. A kit comprising a controlled artificial replicon of a geminivirus as described in any one of claims 1 to 5, or a plant-based directed evolution / screening system as described in any one of claims 6 to 16.

33. The application of genetic element mutants obtained by using the plant-based directed evolution / screening system as described in any one of claims 6 to 16, or by using the plant-based directed evolution / screening system method as described in any one of claims 17 to 19, in plants, particularly in plant genetic engineering.

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