Use of protacs in plant antiviral

Abstract

This invention provides the use of protein degradation-targeting chimeras in plant antiviral or plant breeding applications. This invention also provides a targeted protein degradation platform against the P9-1 protein of rice black-streaked dwarf virus and a method for developing rice black-streaked dwarf virus resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of PROTAC in plant antiviral therapy. Background Technology

[0002] Rice black-streaked dwarf virus (RBSDV) is mainly transmitted in nature by planthoppers (…). Laodelphax striatellus RBSDV spreads in crops through a continuous cycle. When crops are infected with RBSDV, they exhibit symptoms such as short, broad, stiff leaves that turn dark green. As infected plants grow, white waxy protrusions appear on the veins, outer leaf sheaths, and stems. These white protrusions gradually turn dark brown, becoming more prominent. This not only affects the plant's appearance but also significantly impacts crop quality and yield. According to statistics, in areas severely affected by RBSDV outbreaks, rice yield reductions can reach 30%-40%, and in the most severe cases, can lead to total crop failure.

[0003] Currently, the main control methods for RBSDV include developing resistant varieties, screening for resistant genes, and controlling virulent vectors such as the planthopper. However, different crop varieties exhibit significant differences in their resistance to RBSDV, and only a few resistant varieties have been identified so far. The lack of RBSDV-resistant rice and maize varieties increases the difficulty of disease control in agricultural production. RBSDV is mainly spread in nature by planthoppers, whose unique life habits provide a basis for its spread. Screening for resistant genes within the crop itself is quite difficult. Overexpression of existing resistant genes can easily disrupt the crop's metabolic pathways, adversely affecting its growth and development.

[0004] Therefore, there is an urgent need to develop new methods for the prevention and control of RBSDV. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a novel method for crop disease resistance and constructs a new targeted protein degradation platform. This invention enables the specific and efficient degradation of plant target proteins, such as the P9-1 protein in RBSDV, within the plant without damaging the plant's genome, thereby enhancing crop resistance to plant viruses such as rice black-streaked dwarf virus.

[0006] According to one aspect of the invention, the use of protein degradation-targeting chimeras in plant antiviral or plant breeding is provided.

[0007] In some embodiments, the protein degradation targeting chimera includes a target protein binding domain and a target protein degradation domain.

[0008] In some embodiments, the target protein binding domain includes one or more of monomers, antibodies, antibody fragments, scaffold proteins, peptide conjugates, or ligands.

[0009] In some embodiments, the target protein binding domain includes a domain that binds to plant virus proteins.

[0010] In some embodiments, the target protein degradation domain includes ubiquitin ligase (E3) and / or ubiquitin conjugation enzyme (E2).

[0011] In some embodiments, the protein degradation targeting chimera is a bioPROTAC molecule.

[0012] In some embodiments, the target protein binding domain and the degradation domain are directly connected or connected via a linker.

[0013] In some embodiments, the linker is a polypeptide linker.

[0014] In some embodiments, the connector has a general formula (G) n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m Compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, n and m are integers from 1 to 10. Preferably, n is an integer from 1 to 4, and m is an integer from 1 to 3.

[0015] In some embodiments, the plants include crops, wild plants, and artificially cultivated non-crop plants.

[0016] In some embodiments, the crop includes one or more of food crops, cash crops, vegetable crops, fruit crops, and forage crops.

[0017] In some embodiments, the plant target protein includes plant viral proteins.

[0018] In some embodiments, the plant target proteins include one or more of the following: capsid proteins, envelope proteins, nucleocapsid proteins, replication proteins, motility proteins, RNA silencing repressors, and pathogenic proteins of plant viruses.

[0019] In some implementations, protein degradation-targeting chimeras with plant antiviral functions are stably transformed into corresponding plants through tissue culture to achieve plant breeding.

[0020] According to another aspect of the present invention, a polypeptide that specifically binds to the P9-1 protein of rice black-streaked dwarf virus is provided, said polypeptide comprising any one of the amino acid sequences shown in SEQ ID NO: 3-6, or a conserved variant of any one of the amino acid sequences shown in SEQ ID NO: 3-6 obtained by adding, deleting, substituting or modifying one or more amino acids.

[0021] According to another aspect of the present invention, a bioPROTAC molecule is provided, the bioPROTAC molecule comprising a target protein binding domain and a target protein degradation domain, wherein the target protein binding domain comprises the polypeptide described in the present invention.

[0022] In some embodiments, the target protein degradation domain includes ubiquitin ligases and / or ubiquitin-binding enzymes.

[0023] In some embodiments, the ubiquitin ligase includes, but is not limited to, one or more of SPOP (Speckle-type POZ (pox virus and zinc finger) protein), HECT, RING, etc.

[0024] In some embodiments, the ubiquitin-binding enzyme includes, but is not limited to, Ube2A, Ube2B, Ube2D1, UBE2D2, UBE2D3, Ube2D4, or mutants thereof.

[0025] In some embodiments, the target protein binding domain and the degradation domain are directly connected or connected via a linker.

[0026] In some embodiments, the linker is a polypeptide linker.

[0027] In some embodiments, the connector has a general formula (G) n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m Compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, n and m are integers from 1 to 10. Preferably, n is an integer from 1 to 4, and m is an integer from 1 to 3.

[0028] In some implementations, the binding domain of the P9-1 protein is designed as a target protein ligand through the unique mechanism of action of bioPROTAC. It is then linked with an E3 ubiquitin ligase that specifically degrades the P9-1 protein to form a bioPROTAC molecule. This molecule specifically degrades the P9-1 protein in RBSDV in crops, achieving antiviral effects without damaging the original genome.

[0029] According to another aspect of the present invention, an isolated nucleic acid molecule is provided that encodes the polypeptide or the bioPROTAC molecule described herein.

[0030] According to another aspect of the present invention, a recombinant expression vector is provided, which contains the nucleic acid molecule described in the present invention.

[0031] In some embodiments, the recombinant expression vector includes a plant expression vector, preferably a plant binary expression vector.

[0032] According to another aspect of the present invention, a host cell is provided which contains the nucleic acid molecule or the recombinant expression vector described in the present invention, or expresses the polypeptide or the bioPROTAC molecule described in the present invention.

[0033] According to another aspect of the present invention, the use of the polypeptide, the bioPROTAC molecule, the nucleic acid molecule, the recombinant expression vector, or the host cell described herein in plant antiviral or plant breeding is provided.

[0034] In some embodiments, the plant is a grass (Poaceae).

[0035] In some embodiments, the plant includes one or more of rice, corn, wheat, barley, sorghum, and millet.

[0036] In some embodiments, the plant target protein includes plant viral proteins.

[0037] In some embodiments, the plant target protein includes one or more of the following: capsid protein, envelope protein, nucleocapsid protein, replication protein, motility protein, RNA silencing repressor, and pathogenic protein of plant viruses; preferably, the P9-1 protein of rice black-streaked dwarf virus.

[0038] In some embodiments, the plant antiviral activity includes plant resistance to rice black-streaked dwarf virus.

[0039] In some implementations, by analyzing the structure of the RBSDV virus P9-1 protein, a binding protein capable of binding to it is designed and generated. This protein is then linked to an E3 ligase that specifically degrades the P9-1 protein to form a new bioPROTAC. This enables specific recognition and degradation of the P9-1 protein in tobacco and wheat, achieving antiviral effects. This breaks away from the traditional method of crop disease resistance breeding, which requires screening for disease-resistant varieties or genes, greatly increasing the application of crop disease resistance breeding.

[0040] According to another aspect of the present invention, a method for ubiquitination to degrade plant target proteins or to regulate the expression level of plant target proteins is provided, the method comprising the step of contacting a protein degradation targeting chimera with the plant target protein.

[0041] In some embodiments, the plant target protein includes plant viral proteins.

[0042] In some embodiments, the plant target protein includes one or more of the following: capsid protein, envelope protein, nucleocapsid protein, replication protein, motility protein, RNA silencing repressor, and pathogenic protein of plant viruses, preferably the P9-1 protein of rice black-streaked dwarf virus.

[0043] In some embodiments, the protein degradation targeting chimera includes a target protein binding domain and a target protein degradation domain.

[0044] In some embodiments, the target protein binding domain includes one or more of monomers, antibodies, antibody fragments, scaffold proteins, peptide conjugates, or ligands.

[0045] In some embodiments, the target protein binding domain includes a domain that binds to plant virus proteins.

[0046] In some embodiments, the target protein degradation domain includes ubiquitin ligases and / or ubiquitin-binding enzymes.

[0047] In some embodiments, the protein degradation targeting chimera is a bioPROTAC molecule.

[0048] In some embodiments, the binding domain and the degradation domain are directly connected or connected via connectors.

[0049] In some embodiments, the linker is a polypeptide linker.

[0050] In some embodiments, the connector has a general formula (G) n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m Compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, n and m are integers from 1 to 10. Preferably, n is an integer from 1 to 4, and m is an integer from 1 to 3.

[0051] In some embodiments, the protein degradation targeting chimera includes the bioPROTAC molecule described in this invention.

[0052] In some embodiments, the plants include crops, wild plants, and artificially cultivated non-crop plants.

[0053] In some embodiments, the crop includes one or more of food crops, cash crops, vegetable crops, fruit crops, and forage crops.

[0054] In some embodiments, the plant is a grass (Poaceae).

[0055] In some embodiments, the plant includes one or more of rice, corn, wheat, barley, sorghum, and millet.

[0056] According to another aspect of the present invention, a method for plant antiviral therapy is provided, the method comprising the step of causing a protein degradation-targeting chimera to be expressed in the plant.

[0057] In some embodiments, the protein degradation targeting chimera includes a target protein binding domain and a target protein degradation domain.

[0058] In some embodiments, the target protein binding domain includes one or more of monomers, antibodies, antibody fragments, scaffold proteins, peptide conjugates, or ligands.

[0059] In some embodiments, the target protein binding domain includes a domain that binds to plant virus proteins.

[0060] In some embodiments, the target protein degradation domain includes ubiquitin ligases and / or ubiquitin-binding enzymes.

[0061] In some embodiments, the protein degradation targeting chimera is a bioPROTAC molecule.

[0062] In some embodiments, the binding domain and the degradation domain are directly connected or connected via connectors.

[0063] In some embodiments, the linker is a polypeptide linker.

[0064] In some embodiments, the connector has a general formula (G) n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m Compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, n and m are integers from 1 to 10. Preferably, n is an integer from 1 to 4, and m is an integer from 1 to 3.

[0065] In some embodiments, the protein degradation targeting chimera includes the bioPROTAC molecule described in this invention.

[0066] In some embodiments, the plants include crops, wild plants, and artificially cultivated non-crop plants.

[0067] In some embodiments, the crop includes one or more of food crops, cash crops, vegetable crops, fruit crops, and forage crops.

[0068] In some embodiments, the plant is a grass (Poaceae).

[0069] In some embodiments, the plant includes one or more of rice, corn, wheat, barley, sorghum, and millet.

[0070] In some embodiments, the method includes the step of introducing a nucleic acid molecule containing an encoding the protein degradation-targeting chimera into the plant.

[0071] In some embodiments, the method includes a transformation step using an Agrobacterium-mediated method.

[0072] In some implementations, the method includes the following steps: S1: Transform a viral vector containing a nucleic acid molecule encoding the protein degradation targeting chimera into a viral expression host; S2: The transformed viral expression host is transformed into the plant using a virus-specific vector.

[0073] In some embodiments, step S1 includes: transforming the viral vector into Agrobacterium tumefaciens, and then injecting the transformed Agrobacterium tumefaciens into a dicotyledonous plant. In some embodiments, the dicotyledonous plant is a leaf of Nicotiana benthamiana.

[0074] In some embodiments, step S2 includes: converting a virus extracted from the dicotyledonous plant into a planthopper, and then infecting the plant with the planthopper, thereby causing the plant to express the protein degradation-targeting chimera. Attached Figure Description

[0075] Figure 1This paper presents the design of a P9-1 binding binder and its evaluation of binding ability with the P9-1 protein. Specifically, (a) shows potential binding sites on the P9-1 octamer binding surface, with OBP40 as an example, illustrating the binding site of OBP40 with P9-1; (b) shows potential binding sites on the P9-1 dimer binding surface, with DBP17 as an example, illustrating the binding site of DBP17 with P9-1; (c) shows the yeast two-hybrid results of the octamer binding binder (represented by OBP15 and OBP40) interacting with P9-1; (d) shows the yeast two-hybrid results of the dimer binding binder (represented by DBP8 and DBP17) interacting with P9-1; and (e) shows the results of the binding sites of OBP15, OBP40, DBP8, and DBP40 interacting with P9-1. f shows the results of bimolecular fluorescence complementation of binder and P9-1, represented by OBP15, OBP40, DBP8, and DBP17; g shows the results of dual-luciferase of binder and P9-1 after co-transformation of Sf9 cells with binder and P9-1, represented by OBP15, OBP40, DBP8, and DBP17; h shows the diffusion rate of P9-1 aggregates in Sf9 cells after co-transformation of Sf9 cells with binder and P9-1, represented by OBP15, OBP40, DBP8, and DBP17.

[0076] Figure 2 The degradation of P9-1-GFP protein by vhhGFP4-SPOP is illustrated. Specifically, a shows the interaction between vhhGFP4-SPOP and P9-1-GFP; b shows the Western blot results of vhhGFP4-SPOP degradation of P9-1-GFP protein; c shows the fluorescence results of vhhGFP4-SPOP degradation of P9-1-GFP protein; and d shows the quantitative fluorescence results of c.

[0077] Figure 3 The degradation of P9-1-GFP protein by vhhGFP4-RING is illustrated. Specifically, a shows the interaction between vhhGFP4-RING and P9-1-GFP; b shows the Western blot results of vhhGFP4-RING degradation of P9-1-GFP protein; c shows the fluorescence results of vhhGFP4-RING degradation of P9-1-GFP protein; and d shows the quantitative fluorescence results of c.

[0078] Figure 4The degradation of P9-1-GFP protein by OBP15-SPOP is shown. Among them, a) shows the interaction between OBP15-SPOP and P9-1-GFP; b) shows the Western blot results of OBP15-SPOP degradation of P9-1-GFP protein; c) shows the fluorescence experimental results of OBP15-SPOP degradation of P9-1-GFP protein; and d) shows the quantitative fluorescence results of c.

[0079] Figure 5 The degradation of P9-1-GFP protein by OBP40-SPOP is shown. Among them, a shows the interaction between OBP40-SPOP and P9-1-GFP; b shows the Western blot results of OBP40-SPOP degradation of P9-1-GFP protein; c shows the fluorescence experimental results of OBP40-SPOP degradation of P9-1-GFP protein; and d shows the quantitative fluorescence results of c.

[0080] Figure 6 The specificity of OBP15-SPOP and OBP40-SPOP in degrading P9-1 protein is shown. Among them, a shows the Western blot results of GFP after co-injection with vhhGFP4-SPOP, and b shows the Western blot results of GFP after co-injection with OBP15-SPOP or OBP40-SPOP.

[0081] Figure 7 The results show that OBP15-SPOP and OBP40-SPOP induced RBSDV resistance in wheat. Figure a shows the successful expression of the BYSMV vector carrying bioPROTAC in wheat; Figure b shows the Western blot results of the proteins expressed by OBP15-SPOP and OBP40-SPOP in wheat; Figure c shows the quantitative reverse transcription polymerase chain reaction results of the RBSDV coat protein CP gene in wheat carrying bioPROTAC and the control group; Figure d shows the leaf phenotype results of wheat carrying bioPROTAC and the control group after RBSDV infection; Figure e shows the Western blot results of the P10 protein in RBSDV in wheat carrying bioPROTAC and the control group after RBSDV infection. Detailed Implementation

[0082] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0083] The terminology used in one or more embodiments of this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the one or more embodiments of this specification. The singular forms “a,” “described,” and “the” as used in one or more embodiments of this specification and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used in one or more embodiments of this specification refers to and includes any or all possible combinations of one or more associated listed items.

[0084] It should be understood that although the terms first, second, etc., may be used to describe various information in one or more embodiments of this specification, such information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another. For example, first may also be referred to as second without departing from the scope of one or more embodiments of this specification, and similarly, second may also be referred to as first. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to a determination."

[0085] In this invention, the term "multiple" can refer to two or more, and "at least one" can refer to one, two or more.

[0086] The term "and / or" in this invention is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this invention generally indicates that the preceding and following related objects have an "or" relationship.

[0087] The term "linker" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. Domains in the molecules of the present invention can be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. Linkers should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains to which they are linked fold correctly and are properly presented to perform their biological functions. In various embodiments, linkers have a flexible conformation. Suitable flexible linkers include, for example, those containing glycine, glutamine, and / or serine residues. In some embodiments, the amino acid residues in the linker can be arranged in small repeating units of up to five amino acids.

[0088] The term "conservative variant" refers to a protein variant obtained by inserting, extending, substituting, or deleting amino acids in its amino acid sequence without altering or substantially altering its core function and three-dimensional conformation. For example, in this article, "conservative variant" refers to a protein that retains its ability to specifically bind to the rice black-streaked dwarf virus P9-1 protein even after one or more amino acid sequences represented in any of SEQ ID NO: 3-6 have been added, deleted, or substituted.

[0089] The term "substitution" as used herein for amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another different "substituted" amino acid residue. The term "insertion" as used herein for amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. While inserts typically consist of one or two inserted amino acid residues, larger "peptide inserts" can also be prepared, for example, inserts of about three to five or even up to about ten, fifteen, or twenty amino acid residues. As disclosed above, the inserted residues can be naturally occurring or non-naturally occurring. The term "deletion" as used herein for amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

[0090] Variants or fragments thereof of the present invention may contain conserved amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. A “conserved amino acid substitution” is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, in this document, essential or non-essential amino acid residues in mutants are preferably replaced with another amino acid residue from the same side chain family.

[0091] Ubiquitination is a highly conserved post-translational modification of proteins in eukaryotes, referring to the covalent binding of ubiquitin molecules to target proteins through a series of enzymes, thereby regulating protein function. After years of research, thousands of proteins have been identified as involved in ubiquitin degradation. The ubiquitin-proteasome system (UPS) is the main pathway for intracellular protein degradation, participating in more than 80% of protein degradation in cells. UPS is a multi-step reaction process involving various proteins, including ubiquitin (Ub), ubiquitin activator E1, ubiquitin conjugate E2, ubiquitin-protein ligase E3, proteasome 26S, and ubiquitin dissociators (DUBs). E1 is responsible for activating Ub molecules and forming high-energy linkages, initiating the ubiquitination process. E2 transfers the activated Ub from E1 and, with the assistance of E3 (under natural conditions), covalently links the Ub to the substrate protein. E3 recognizes the substrate protein and catalyzes the transfer of the Ub carried by E2 to the substrate, achieving substrate-specific ubiquitination.

[0092] Proteolysis-targeting chimeras (PROTACs) are small molecule compounds designed based on the natural ubiquitin-proteasome system (UPS) within cells. They cleverly utilize the small molecule chimera to simultaneously recruit the target protein and an E3 ubiquitin ligase, inducing ubiquitination of the target protein, which is then degraded by the proteasome, achieving "catalytic degradation" of the target protein. Small molecule PROTACs utilize a limited range of E3 enzymes; currently, only a dozen or so small molecule ligands of E3 enzymes have been reported. BioPROTACs are similar to small molecule PROTACs, but they use complete E3 ligases or E3 ligase subunits to ligate the target protein, allowing the use of any E3 ligase. Compared to PROTACs, E3 has greater affinity and specificity for the target protein, and the enhanced diversity of E3 ligases enables bioPROTACs to achieve highly efficient degradation. The unique mechanism of action of bioPROTACs allows them to overcome the limitations of traditional small molecule inhibitors, fundamentally changing the approach to targeting "undruggable" proteins that traditional small molecules cannot effectively inhibit, and greatly expanding the target range for drug development. A typical bioPROTAC molecule consists of three key parts: a ligand that specifically targets the protein of interest (POI), an E3 ubiquitin ligase, and a linker between the two ligands. Each of these three parts plays a very unique and important role, while also cooperating and complementing each other to achieve the specific degradation of the target protein.

[0093] Plant virus proteins are a collective term for proteins encoded by the plant virus genome that play crucial roles in viral infection, replication, transmission, and pathogenesis. Based on function, they can be divided into two main categories: structural proteins and non-structural proteins. Structural proteins constitute the outer shell or core components of the virus particle, responsible for protecting the viral nucleic acid and mediating viral entry into the host cell. These mainly include capsid proteins (CP), envelope proteins (EP), and nucleocapsid proteins (N). Non-structural proteins do not participate in virus particle assembly but are indispensable in viral replication, proliferation, and pathogenesis. These mainly include replication-related proteins, motility proteins, RNA silencing repressors, and pathogenicity-related proteins.

[0094] Rice black-streaked dwarf virus (RBSDV) belongs to the subgroup II of the genus Fijidisease virus in the family Reoviridae. Its primary hosts are gramineous crops such as rice, maize, and wheat. RBSDV viral particles consist of inner and outer capsids, exhibiting a typical icosahedral spherical structure. The double capsid contains spikes and has a diameter of approximately 75-80 nm. This structure provides the viral particles with good stability, facilitating their survival and spread in nature. RBSDV is a double-stranded RNA (dsRNA) virus with a genome length of 29,141 bp, composed of 10 dsRNAs, named S1-S10 based on their migration speed on gel electrophoresis. The P9-1 protein, encoded by S9 in the RBSDV genome, participates in virion assembly, used for viral genome replication and progeny viral particle assembly. The P10 protein encoded by S10 in the RBSDV genome is the RBSDV coat protein, or CP (Coat Protein), which is the main structural component of the outer capsid of the viral particle and is responsible for core functions such as genome protection, host recognition, and infection.

[0095] Barley yellow striate mosaic virus (BYSMV) is a plant rhabdovirus that is mainly transmitted by planthoppers and can infect a variety of grass crops. It has been modified into a plant virus expression vector.

[0096] In the early stages of rice black-streaked virus infection, the P9-1 protein first forms inclusion bodies in the host cell, participating in virion assembly and providing a suitable environment for viral genome replication and progeny virus particle assembly. Studies have shown that in cells, P9-1 first forms dimers, and then each dimer extends an arm (C arm) to both sides, binding with adjacent dimers through their arms and shoulders to form a cylindrical octamer. The P9-1 dimer is the basic building block of the octamer.

[0097] Based on the crucial role of the P9-1 protein in RBSDV viral assembly, this invention designs a binding protein targeting the P9-1 protein and screens for E3 ubiquitin ligases that specifically recognize the P9-1 protein. These ligases are then combined with the designed binding protein to construct a novel bioPROTAC molecule. This molecule achieves efficient degradation of the P9-1 protein within the crop without disrupting the crop's genome, thereby enhancing the crop's resistance to rice black-streaked dwarf virus. Therefore, bioPROTAC possesses the fundamental ability to degrade RBSDV viral proteins for disease resistance.

[0098] In some implementations, a protein design method developed in David Baker's lab is used to design binding proteins targeting the P9-1 protein. Specifically, based on the structural features of the binding interfaces between the octamer and dimer of the P9-1 protein, an initial structural design is generated using RFdiffusion technology (Watson, JL, Juergens, D., Bennett, NR et al. De novodesign of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023).), followed by sequence optimization using ProteinMPNN (J. Dauparas et al., Robustdeep learning–based protein sequence design using ProteinMPNN. Science 378, 49-56 (2022).). All designed sequences were systematically evaluated using AlphaFold2 (Bennett, NR, Coventry, B., Goreshnik, I. et al. Improving de novo protein binder design with deep learning. Nat Commun 14, 2625 (2023).) and MaSIF (Gainza, P., Sverrisson, F., Monti, F. et al. Deciphering interaction fingerprints from protein molecularsurfaces using geometric deep learning. Nat Methods 17, 184–192 (2020).). OBP15, OBP40, DBP8, and DBP17 showed good P9-1 interaction performance. This result indicates that protein design has considerable potential in protein-protein interaction analysis.

[0099] In some embodiments, this invention successfully established a bioPROTAC-mediated protein degradation system based on vhhGFP4-SPOP and vhhGFP4-RING in tobacco leaves, achieving efficient and specific degradation of the P9-1-GFP protein. Simultaneously, this invention also achieved OBP15-SPOP and OBP40-SPOP-mediated specific degradation of P9-1-GFP in tobacco leaves, providing a reliable technical platform for targeted protein degradation research in plant systems. This invention found that the OBP15-SPOP and OBP40-SPOP-mediated specific degradation of P9-1-GFP depends on the recognition and targeting of the P9-1 protein by OBP15 and OBP40, rather than the GFP protein itself. This result indicates that protein design has considerable potential in terms of protein targeting specificity.

[0100] Furthermore, the bioPROTAC molecules OBP15-SPOP and OBP40-SPOP constructed in this invention achieved degradation of the P9-1 protein in wheat, significantly enhancing the crop's resistance to RBSDV. This is the first time that bioPROTAC molecules have been used for antiviral research in crops, providing proof of concept for the application of bioPROTAC technology in the field of crop antiviral research.

[0101] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0102] Materials and Methods 1. Evaluation of the binding ability of the design binding protein (binder) to P9-1 in Sf9 cells, yeast cells, and tobacco leaves. (1) Design the binder sequence High-affinity target protein ligands can form more stable complexes with target proteins, thereby significantly improving the degradation efficiency of bioPROTAC on target proteins. High-specificity target protein ligands can effectively reduce the off-target effects of bioPROTAC, allowing it to precisely target the protein and further enhancing the effectiveness and selectivity of bioPROTAC. To design binding proteins that specifically recognize P9-1, this invention uses RFdiffusion (RFDiffusion... RoseTTAFold DiffusionThe model takes the atomic coordinates of amino acid residues 194 to 277 of the 3vjj A chain (Uniprot ID: Q913E4) as input, designating residues 222, 229, and 268 as hotspots; and the atomic coordinates of amino acid residues 178 to 238 and 245 to 277 of the 5eft B chain (Uniprot ID: B6SCH3) as input, designing the backbone structure of the binding protein. The ProteinMPNN (Protein Message Passing Neural Network) model is then used to generate amino acid sequences for each backbone structure, yielding the complex structure of the binding protein and the target protein. Then, binding proteins were computationally screened using two methods: the AlphaFold2 initial guess program was run to screen for binding proteins with a plddt value greater than 70 and a PAE value less than 10 for the binding protein-target protein complex structure; the MaSIF-search program was run to screen for binding proteins that matched the surface features of the target protein. The differences in binding ability between the protein-designed binder and the P9-1 protein were also evaluated.

[0103] (2) Sequence acquisition and vector construction The P9-1 sequence and the designed binder sequence were optimized according to the tobacco codon and synthesized by Suzhou Junji Gene Technology Co., Ltd. Subsequently, the P9-1 sequence was cloned into the Sf9 cell (fall armyworm ovary cells) expression vector POPIE-RFP, yeast expression vectors pNC-GADT7 and pNC-GBKT7, dual-luciferase reporter vectors pNC-NLUC and pNC-CLUC, and bimolecular fluorescent complementary reporter vectors pNC-Ecnhe and pNC-Enn, respectively. The designed binder sequence was cloned into the Sf9 cell expression vector POPIE-GFP, yeast expression vector pNC-GADT7, dual-luciferase reporter vector pNC-NLUC, and bimolecular fluorescent complementary reporter vector pNC-Ecn, respectively. All plasmids were constructed and amplified using *E. coli* DH5α strain (DL1001, Shanghai Weidi Biotechnology Co., Ltd.). The obtained expected products were verified to be correct by Sanger sequencing and are ready for subsequent experiments.

[0104] (3) Yeast dual hybrid Transfer an appropriate amount of carrier DNA (10 mg / mL) into a sterile 1.5 mL EP tube (10 μL per transformation), denature at 100℃ for 5 min, and immediately cool on ice. Repeat once. Take 100 μL of Y2HGold yeast competent cells stored at -80℃, thaw on ice, and under sterile conditions, add 10 μL of carrier DNA, 1-2 μg of pre-chilled target plasmids (pNC-GADT7 and pNC-GBKT7), and 500 μL of PEG3350 / LiAc, and mix by pipetting. Incubate at 30℃ for 30 min, inverting 6-8 times every 15 min. Transfer to a 42℃ water bath for 15 min, inverting 6-8 times every 7.5 min. Remove the EP tube, centrifuge at 5000 r for 1 min at room temperature, and discard the supernatant. Resuspend the yeast cells in 400 μL ddH2O, mix by pipetting, centrifuge at 5000 r for 1 min at room temperature, and discard the supernatant. Yeast cells were resuspended in 100 μL ddH2O and plated onto a two-notch yeast plate (SD-Trp-Leu). The plates were then incubated upside down at 30°C for 2-3 days. Once yeast cells had grown on the two-notch plates, single colonies were selected and dissolved in 10 μL ddH2O. 3 μL of this solution was then spotted onto a four-notch yeast plate (SD-Trp-Leu-Ade-His), with a concentration of 10 μL. 0 Dissolve 1 μL in 9 μL of ddH2O, and spot 3 μL onto a four-slot plate. The result is 10. -1 Dissolve 1 μL in 9 μL of ddH2O, and spot 3 μL onto a four-slot plate. The result is 10. -2 Dissolve 1 μL in 9 μL of ddH2O, and spot 3 μL onto a four-slot plate. The result is 10. -3 Incubate at 30℃ upside down for 2 days.

[0105] (4) Bimolecular fluorescence complementary (BiFC) The constructed vector was transformed into Agrobacterium GV3101, and single colonies were picked to identify positive colonies. The colonies were then treated with 1 mL of Kanamycin-containing antibiotics. Cultured in LB liquid medium with + Rif. Then transferred to 10 mL of double antibiotic (Kana) In Rif) LB liquid medium, incubate at 28°C and 200 rpm for 12-16 h, until the bacterial concentration reaches OD500. 600 The concentration was 1.2-1.5. The bacterial cells were collected by centrifugation and resuspended in 10 mM MgCl2 until the OD of the bacterial culture was reached. 600 The concentration was 1.6. The bacterial cultures were mixed at a 1:1 ratio of pNC-Ecn-A protein to pNC-Enn-B protein until the final concentration of both cultures was OD. 600= 0.8, injected into tobacco. Using pNC-Ecn + pNC-Enn, pNC-Ecn + pNC-Enn-B protein, and pNC-Ecn-A protein + pNC-Enn as negative controls, Agrobacterium was injected into each, and cultured for 3 days. A portion of the injected tobacco leaf was taken using a 10mm punch and placed on a glass slide, ddH2O was added, a coverslip was placed on top, and YFP fluorescence was observed under a laser confocal microscope, with the results recorded simultaneously.

[0106] (5) Luciferase complementarity (LUC) Agrobacterium was treated using the same method as in BiFC.

[0107] Mix pNC-CLUC-A protein and pNC-NLUC-B protein bacterial cultures at a 1:1 ratio until the final concentration of both cultures is OD. 600 =0.8, injected into tobacco. Using pNC-CLUC + pNC-NLUC, pNC-CLUC + pNC-NLUC-B protein, and pNC-CLUC-A protein + pNC-NLUC as negative controls, Agrobacterium was injected into each strain and cultured for 3 days. A 1x D-luciferin potassium salt solution was prepared and injected into the same injection site as 3 days prior. The mixture was incubated in the dark for 10 min, and luciferase activity (luminescence value) was measured using a luciferase detector, with images recorded simultaneously.

[0108] (6) SF9 cell transfection First, perform cell passage. Place the cell culture flask horizontally with the opening facing upwards, and add 4 mL of cell culture medium (SF-900Ⅲ SFM cell culture medium diluted 1:100 with penicillin and streptomycin antibiotics, and 1:10 with fetal bovine serum), covering the bottom of the flask. Prepare the cells for transformation the day before transformation. After 3 days of passage, gently pipette the healthy cells into the culture medium, add fresh cell culture medium to 12 mL, and mix well. Add 500 μL of fresh cell culture medium and 500 μL of cell culture solution to a 12-well plate, mix well, and incubate at 25°C. Mix 1 μg of the plasmid to be transfected and 200 μL of fresh cell culture medium in a 1.5 mL EP tube, add 10 μL of X-tremeGENE™ HP DNA transfection reagent to each tube, shake well, and let stand for 10-15 min. Add the mixture to the wells of the 12-well plate, avoiding suspension of adherent cells, and immediately agitate after addition. After adding all the ingredients, tighten the 12-well plate cap, wrap it tightly with sealing film, and incubate at 25°C for 24-48 hours.

[0109] For slide preparation and observation, place coverslips in a small dish. You can pre-spread concanavalin A solution on the coverslips and incubate for one minute, then remove the concanavalin A solution. Place the coverslips in a fume hood to dry quickly; a white protein residue will appear on the coverslip after drying. Resuspend cells transfected with plasmids (cultured for 24-48 h) in the wells by pipetting and mixing, then completely cover the coverslips and incubate for 15-30 min to allow the cells to adhere. Aspirate the cell culture medium, but do not remove the cells. Cover the cells with 4% cell fixative and incubate for 15-20 min, then aspirate the cell fixative. Cover the cells with 1xPBS and incubate for 5 min; repeat three times. Add 20 μL of mounting medium to a glass slide, use tweezers to lift the coverslip, and blot dry with absorbent paper. Place the coverslip, cell side down, onto the glass slide slowly to avoid creating air bubbles. After covering the slide, do not move it to avoid damaging the cells. Apply nail polish to the four corners of the slide to secure it. After preparation, store the slide at 4°C away from light. Alternatively, you can observe the results directly under a confocal laser microscope and take photos simultaneously.

[0110] 2. Screening for E3 ubiquitin ligases from tobacco leaves that can specifically degrade P9-1 protein. The E3 ubiquitin ligase family is large and poorly conserved across species, with each E3 exhibiting strong specificity in selecting substrate ubiquitination sites. To screen for E3 ubiquitin ligases capable of specifically degrading P9-1 protein, this invention uses the reported high-affinity anti-GFP nanobody vhhGFP4 as the target protein ligand. Using an Agrobacterium-mediated transient transformation method, vhhGFP4-E3 and P9-1-GFP were co-transformed into Nicotiana benthamiana leaves. The degradation efficiency of different E3 enzymes on P9-1 protein was evaluated using GFP fluorescence intensity detection and Western blotting analysis.

[0111] (1) Sequence acquisition and vector construction Candidate E3 ubiquitin ligases and the P9-1 (Uniprot ID: Q913E4) sequence were optimized according to the tobacco codon and synthesized by Suzhou Junji Gene Technology Co., Ltd. Subsequently, using the ClonExpress Ultra One StepCloning Kit (C115-01, Nanjing Novizan Biotechnology Co., Ltd.), the synthesized E3 ubiquitin ligase was cloned into the plant binary expression vector pCambia1300-3xFLAG-VhhGFP4, and fused with it at the C-terminus of VhhGFP4 via a linker (GS) for expression. The P9-1 target protein sequence was ligated into the plant binary expression vector pEarleyGate104, placed between the 35S promoter and the OCS terminator, and fused with GFP for expression. A 3xFLAG protein tag was added to the N-terminus of the sequence for subsequent detection of protein expression levels. All plasmids were constructed and amplified using *Escherichia coli* DH5α strain (DL1001, Shanghai Weidi Biotechnology Co., Ltd.). The obtained expected construct, after being verified as correct by Sanger sequencing, was transformed into Agrobacterium GV3101 strain (AC1001, Shanghai Weidi Biotechnology Co., Ltd.) for use in subsequent experiments.

[0112] (2) Transient expression test of tobacco Single colonies of Agrobacterium, identified by PCR, were picked and incubated overnight at 28°C and 200 rpm in 1 mL LB liquid medium (Kana + Rif, working concentration of Kana 50 mg / L, working concentration of Rif 40 mg / L). 200 μL of the incubator was added to 10 mL LB liquid medium (Kana + Rif) and incubated at 28°C and 200 rpm for 12-16 h until the bacterial concentration reached OD600 = 1.2-1.5. The colonies were collected at 3000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in 10 mM MgCl2 to the specified OD value, and MES (final concentration 10 mM) and AS (final concentration 0.2 mM) were added. The cells were then incubated in the dark for 2 h. The cells were then injected into tobacco leaves (two 12 mm² injection sites per plant). 2 The mixture consists of two circles (approximately 0.5 ml in total volume). Each group is injected with three tobacco plants, each with three leaves, and cultured according to experimental requirements.

[0113] (3) Western blot Samples were taken 96 hours after injection. Two discs were taken from each sample using a 10mm punch, ground, and then 2x SDS loading buffer was added. The mixture was incubated on ice for 10 minutes, then boiled at 100°C for 10 minutes. After cooling, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected for loading. Electrophoresis was performed using a 10% SDS-PAGE gel. The proteins were then transferred to a 0.45µm PVDF membrane, stained with Ponceau S (P8330, Beijing Solarbio Science & Technology Co., Ltd.), and residual dye was washed away with TBST. The membrane was blocked with 5% skim milk powder for 1 hour, washed with TBST, and then anti-Flag-Tag mAb (AE005, Abiotech Co., Ltd.) diluted 1:20,000 in 3% BSA solution was added. The membrane was incubated overnight at 4°C. After primary antibody treatment, the membrane was washed and incubated with a 1:5000 dilution of HRP-conjugated Goat anti-Mouse IgG (H+L) (AS003, Abiotech Biotechnology Co., Ltd.) secondary antibody at room temperature for 1 hour. Unbound antibodies were then washed away, and signal detection was performed using a Clarity Western ELISA kit (1705060; Bio-Rad), with imaging using a Tanon-4600 chemiluminescence imaging system. The 45S large subunit band, stained with Ponceau S, was used as an internal control to assess the degradation of the target protein.

[0114] (4) Fluorescence observation This experiment was conducted on the public instrument platform of the College of Agriculture, China Agricultural University, using a Zeiss LSM880 inverted confocal microscope. Ar lasers (458nm, 488nm, 514nm) were used; for the 488nm laser, the laser power was uniformly set to 0.6%–2.4%. Six fields of view were randomly selected for each sample, and imaging was performed under a 10× objective lens (NA 0.45). The laser intensity was kept consistent throughout the observation process to ensure comparability of the results.

[0115] 3. The screened specific E3 ubiquitin ligase was combined with the designed target protein ligand to form a new bioPROTAC molecule, and the degradation efficiency of the bioPROTAC molecule on P9-1 protein was evaluated.

[0116] (1) Sequence acquisition and vector construction The E3 ubiquitin ligase, binder sequence, and P9-1 protein sequence were optimized according to the tobacco codon and synthesized by Suzhou Junji Gene Technology Co., Ltd. Subsequently, using the ClonExpress Ultra One Step Cloning Kit (C115-01, Nanjing Novizan Biotechnology Co., Ltd.), the binder and E3 ubiquitin ligase were first recombined, with a GS amino acid sequence added as a linker. The recombinant product was cloned into the plant binary expression vector pCambia1300-3xFLAG and fused with it at the C-terminus. The P9-1 target protein sequence was ligated into the plant binary expression vector pEarleyGate104, placed between the 35S promoter and the OCS terminator, and fused with GFP for expression. A 3xFLAG protein tag was added to the N-terminus of the sequence for subsequent detection of protein expression levels. All plasmids were constructed and amplified using *E. coli* DH5α strain (DL1001, Shanghai Weidi Biotechnology Co., Ltd.). The obtained expected construct, after being verified as correct by Sanger sequencing, was transformed into Agrobacterium GV3101 strain (AC1001, Shanghai Weidi Biotechnology Co., Ltd.) for use in subsequent experiments.

[0117] (2) Transient expression test of tobacco Single colonies of Agrobacterium, identified by PCR, were picked and incubated overnight at 28°C and 200 rpm in 1 mL LB liquid medium (Kana + Rif, working concentration of Kana 50 mg / L, working concentration of Rif 40 mg / L). 200 μL of the incubator was added to 10 mL LB liquid medium (Kana + Rif) and incubated at 28°C and 200 rpm for 12-16 h until the bacterial concentration reached OD600 = 1.2-1.5. The colonies were collected at 3000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in 10 mM MgCl2 to the specified OD value, and MES (final concentration 10 mM) and AS (final concentration 0.2 mM) were added. The cells were then incubated in the dark for 2 h. The cells were then injected into tobacco leaves (two 12 mm² injection sites per plant). 2 The mixture consists of two circles (approximately 0.5 ml in total volume). Each group is injected with three tobacco plants, each with three leaves, and cultured according to experimental requirements.

[0118] (3) Western blot Samples were taken 96 hours after injection. Two discs were taken from each sample using a 10mm punch, ground, and then 2x SDS loading buffer was added. The mixture was incubated on ice for 10 minutes, then boiled at 100°C for 10 minutes. After cooling, the mixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was collected for loading. Electrophoresis was performed using a 10% SDS-PAGE gel. The proteins were then transferred to a 0.45µm PVDF membrane, stained with Ponceau S (P8330, Beijing Solarbio Science & Technology Co., Ltd.), and residual dye was washed away with TBST. The membrane was blocked with 5% skim milk powder for 1 hour, washed with TBST, and then anti-Flag-Tag mAb (AE005, Abiotech Co., Ltd.) diluted 1:20,000 in 3% BSA solution was added. The membrane was incubated overnight at 4°C. After primary antibody treatment, the membrane was washed and incubated with a 1:5000 dilution of HRP-conjugated Goat anti-Mouse IgG (H+L) (AS003, Abiotech Biotechnology Co., Ltd.) secondary antibody at room temperature for 1 hour. Unbound antibodies were then washed away, and signal detection was performed using a Clarity Western ELISA kit (1705060; Bio-Rad), with imaging using a Tanon-4600 chemiluminescence imaging system. The 45S large subunit band, stained with Ponceau S, was used as an internal control to assess the degradation of the target protein.

[0119] (4) Fluorescence observation This experiment was conducted on the public instrument platform of the College of Agriculture, China Agricultural University, using a Zeiss LSM880 inverted confocal microscope. Ar lasers (458nm, 488nm, 514nm) were used; for the 488nm laser, the laser power was uniformly set to 0.6%–2.4%. Six fields of view were randomly selected for each sample, and imaging was performed under a 10× objective lens (NA 0.45). The laser intensity was kept consistent throughout the observation process to ensure comparability of the results.

[0120] 4. bioPROTAC molecules degrade P9-1 protein and resist RBSDV in crops. (1) Sequence acquisition and vector construction Using the ClonExpress Ultra One Step Cloning Kit (C115-01, Nanjing Novizan Biotechnology Co., Ltd.), binder and E3 ubiquitin ligase were first used for fragment recombination. A GS amino acid sequence was added as a linker in the middle, and the recombinant product was cloned into the BYSMV expression vector BYSMV-GR, positioned between the N and P proteins, replacing the RFP protein, and fused with the GFP protein for expression. A 3xFLAG protein tag was added to the N-terminus of the sequence for subsequent protein expression level detection. All plasmids were constructed and amplified using *E. coli* DH5α strain (DL1001, Shanghai Weidi Biotechnology Co., Ltd.). After Sanger sequencing verification, the obtained expected products were transformed into *Agrobacterium* GV3101 strain (AC1001, Shanghai Weidi Biotechnology Co., Ltd.) for subsequent experiments.

[0121] (2) Using the BYSMV reverse operation platform to convert wheat The BYSMV expression vector was used to inoculate planthoppers. Agrobacterium carrying the BYSMV virus plasmid was injected into Nicotiana benthamiana. Thirteen days later, tobacco leaves showing significant fluorescence (indicating high infection rates) were selected under a fluorescent hammer, and the non-injected areas of the leaves were cut off. Virus extraction buffer was added at a 1:2 ratio, and the tobacco leaves were ground into a viscous consistency in a pre-chilled mortar on ice. The mixture was then transferred to a 1.5 mL EP tube. The tube was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. The supernatant was then temporarily stored on ice.

[0122] Planthopper larvae, approximately 12 days old, were blown into 50 mL centrifuge tubes and placed on ice to freeze and stun them (approximately 30 minutes). The glass capillary tube was then drawn into a fine needle using a needle puller and cut into an injection needle shape under a stereomicroscope. The microinjection needle was inserted into the micromanipulator, and the distance between the micromanipulator and the stereomicroscope stage was adjusted. The previously stored tobacco juice was drawn into the microinjection needle. Five to six stunned planthoppers were collected and scattered on an antibiotic-free culture medium plate, placed under the stereomicroscope objective. The insects were first located in the eyepiece field of view, and the microinjection needle was moved until the needle tip was visible in the lens. The culture medium was moved to position the insects directly under the needle. The micromanipulator was then used to insert the needle into the thorax cavity of the insect, injecting 13.8 mL of tobacco juice. The injected insects were then placed in new rice seedlings, and the process was repeated. Each group of bacterial solutions was injected with approximately 200 worms. After injection, the worms were cultured for about 10 days until a fluorescent hammer was used to observe obvious fluorescence within the worms.

[0123] Inject the venomous insects into crops such as wheat, allowing them to infect for 4-7 days (4 days in summer). Observe the infection status using a fluorescent hammer. Remove the venomous insects and cultivate the infected crops as needed, observing the crop phenotypes.

[0124] (3) Inoculation with RBSDV virus Planthoppers carrying the RBSDV virus were blown into wheat seedlings carrying bioPROTAC (binder-E3) to infect them with the RBSDV virus. The infected planthoppers were blown out after about 4-7 days (4 days in summer). Wheat seedlings were used as a control to observe the RBSDV infection status of wheat.

[0125] (4) Western blot Sampling was performed 15-20 days after infection, with each sample consisting of 3-4 cm leaf fragments. The fragments were ground and then added to 2x SDS loading buffer. After standing on ice for 10 min, the protein was boiled at 100℃ for 10 min, cooled, and centrifuged at 12000 rpm for 5 min. The supernatant was then used for sample loading. Electrophoresis was performed using a 10% SDS-PAGE gel. The protein was then transferred to a 0.45 μm PVDF membrane, stained with Ponceau S (P8330, Beijing Solarbio Science & Technology Co., Ltd.), and residual dye was washed away with TBST. The membrane was blocked with 5% skim milk powder for 1 h, washed with TBST, and then anti-P10 mAb (diluted 1:5000 in 3% BSA solution) was added. The membrane was incubated overnight at 4℃. After primary antibody treatment, the membrane was washed and incubated with a 1:5000 dilution of HRP-conjugated Goat anti-Mouse IgG (H+L) (AS003, Abiotech Biotechnology Co., Ltd.) secondary antibody at room temperature for 1 hour. Unbound antibodies were then washed away, and signal detection was performed using a Clarity Western ELISA kit (1705060; Bio-Rad), with imaging using a Tanon-4600 chemiluminescence imaging system. The 45S large subunit band, stained with Ponceau S, was used as an internal control to assess the degradation of the target protein.

[0126] (5) Extraction, transcription and qRT-PCR of total RNA from plant tissues Take an appropriate amount of wheat leaf tissue and grind it with liquid nitrogen. Extract total RNA using the FastPure Universal PlantTotal RNA Isolation Kit (RC411-01, Nanjing Novizan Biotechnology Co., Ltd.). Finally, elute the RNA with 30 μL of RNase-free ddH2O, measure the concentration using Nanodrop, and immediately reverse transcribe or store at -80℃. Based on the measured RNA concentration, take 2 μg of total RNA into an RNase-free 200 μL PCR tube, and add ddH2O to a final volume of 15 μL. Add 1 μL of Enzyme Mix and 4 μL of 5xAll-in-one qRT SuperMix (R333, Nanjing Novizan Biotechnology Co., Ltd.), mix well, and incubate at 50℃ for 15 min, followed by enzyme inactivation at 85℃ for 5 s. Dilute with 180 μL of ddH2O and store at -20℃ as a template for subsequent qRT-PCR.

[0127] Real-time quantitative PCR (qRT-PCR) reactions were performed using a SYBR mixed enzyme. The reaction mixture consisted of 10 μL of 2xSYBR qPCR Master Mix, 1 μL of cDNA template, 0.5 μL of primer, and 8.5 μL of ddH2O. The reaction was conducted on a CFX96 real-time PCR instrument. The reaction program was: pre-denaturation at 95℃ for 30 s, 40 amplification cycles, 95℃ for 10 s, annealing at 60℃ for 20 s, melting curve analysis, and gradual temperature increase from 65℃ to 95℃, detecting changes in fluorescence signal. Sample cDNA was used as a template to amplify each gene requiring quantification, and melting curves for each reference gene were obtained. Three biological replicates and three technical replicates were performed. Template-free controls and reverse transcription negative controls were used to detect potential reagent and genomic DNA contamination. Cq values ​​were obtained for each sample, and the raw Cq values ​​were entered into Microsoft Excel 2016. -ΔΔCt The expression level of the CP gene was calculated using a method.

[0128] The sequence information involved in the above "Materials and Methods" and the following examples is shown in Table 1 below.

[0129] Table 1 Example 1: Design of P9-1 binding binder and evaluation of its binding ability with P9-1 protein As described in Section 1 of "Materials and Methods", in order to design a binder that specifically binds to P9-1, the structural characteristics of the binding interface between the P9-1 protein octamer and dimer were considered (see Section 1). Figure 1(a) and (b) First, the initial structural design was generated using RFdiffusion technology, followed by sequence optimization using ProteinMPNN. All designed sequences were evaluated using the AlphaFold2 and MaSIF-seed systems. To evaluate the binding ability of the designed protein binder to P9-1, yeast double-hybrid, bimolecular fluorescence complementation, and luciferase complementation experiments were used to assess the binding ability of the binder to P9-1 protein. PopIE-P9-1-RFP and PopIE-binder-GFP vectors were co-transfected into Sf9 cells, and their interaction was assessed by observing the fluorescence distribution of GFP and RFP. The diffusion rate of P9-1 protein (i.e., the percentage of cells with diffuse P9-1 protein out of the total number of cells) was calculated by counting the number of cells in which P9-1 protein did not form aggregates.

[0130] Figure 1 The results of c-1f showed that OBP15, OBP40, DBP8 and DBP17 exhibited significant P9-1 binding ability in yeast cells and tobacco cells, with their interaction effects being particularly prominent. Figure 1 The results showed that when P9-1-RFP protein was expressed alone in Sf9 cells, obvious viral inclusion body structures were observed, and P9-1-RFP protein formed significant aggregate structures. Figure 1 The results showed that when P9-1-RFP was co-expressed with OBP15, OBP40, DBP8, and DBP17, P9-1-RFP was diffusely observed in Sf9 cells in multiple fields of view, indicating that the formation of P9-1 aggregates was significantly inhibited. These results suggest that OBP15, OBP40, DBP8, and DBP17 can effectively interfere with the formation of P9-1 aggregates.

[0131] Example 2: vhhGFP4-SPOP and vhhGFP4-RING degrade P9-1-GFP protein E3 ubiquitin ligase is a key component of bioPROTAC. It is responsible for recruiting E2 enzymes to the vicinity of target proteins, recognizing the target proteins through bioPROTAC, performing ubiquitination labeling, and initiating protein ubiquitin degradation signals.

[0132] As described in Section 2 of "Materials and Methods," to screen E3 ubiquitin ligases that can effectively mediate the degradation of P9-1-GFP protein, P9-1-GFP was used as the target protein and co-transformed with different vhhGFP4-E3 vectors into *Nicotiana benthamiana* leaves to evaluate the degradation efficiency of each E3 vector for P9-1-GFP. OD was prepared separately. 600 Agrobacterium vhhGFP4-E3 bacterial suspensions with values ​​of 0.1, 0.5, and 1.0 were used, with the vhhGFP4 vector serving as a control. The results were compared with OD...600 An equal volume of P9-1-GFP Agrobacterium with a concentration of 1.0 was injected into leaves of *Nicotiana benthamiana*. Four days after transformation, changes in GFP fluorescence intensity were observed. GFP fluorescence intensity was statistically analyzed in six fields for each treatment group. Total protein was then extracted for Western blotting analysis. Wild-type (WT) tobacco was used as a negative control. Changes in P9-1-GFP protein expression levels were detected using a Flag-specific antibody to assess the protein degradation efficiency of each E3 ligand.

[0133] This embodiment screened E3 ubiquitin ligases SPOP and RING in tobacco leaves that can specifically degrade the P9-1 protein. Specifically, Figure 2 and Figure 3 The results showed that, 4 days after injection, neither the expression level of P9-1 protein nor the fluorescence intensity of GFP changed significantly in the experimental group injected with P9-1-GFP alone nor in the control group co-injected with P9-1-GFP and vhhGFP4. However, in the experimental group co-injected with P9-1-GFP and vhhGFP4-SPOP or vhhGFP4-RING, the fluorescence intensity of GFP showed a significant dose-dependent decrease with increasing concentration of vhhGFP4-SPOP or vhhGFP4-RING (see [link to study]. Figure 2 c-2d, Figure 3 c-3d). Western blot analysis further confirmed this result: compared with the control group, the expression level of P9-1-GFP in the vhhGFP4-SPOP and vhhGFP4-RING experimental groups was significantly decreased (see c-3d). Figure 2 b、 Figure 3 b). Further protein structure analysis revealed the interaction mechanism between vhhGFP4-SPOP, vhhGFP4-RING, and P9-1-GFP (see [link]). Figure 2 a, Figure 3 a).

[0134] Example 3: OBP15-SPOP degrades P9-1-GFP protein, and OBP40-SPOP degrades P9-1-GFP protein. As described in Section 3 of "Materials and Methods," a bioPROTAC vector containing binder-SPOP was constructed to screen bioPROTAC molecules capable of efficiently and specifically degrading native P9-1 protein. P9-1-GFP was used as the target protein and co-transformed with different binder-SPOP vectors into *Nicotiana benthamiana* leaves to evaluate the degradation efficiency of each bioPROTAC on P9-1-GFP. *Agrobacterium benthamiana* bacterial suspensions with OD600 values ​​of 0.1, 0.5, and 1.0 were prepared, with the binder vector serving as a control. These suspensions were mixed with an equal volume of *Agrobacterium benthamiana* P9-1-GFP containing a P9-1-GFP suspension with an OD600 value of 1.0 and injected into *Nicotiana benthamiana* leaves. Four days after transformation, changes in GFP fluorescence signal intensity were observed. GFP fluorescence intensity was statistically analyzed in six fields for each treatment group, followed by total protein extraction for Western blotting analysis. Wild-type (WT) tobacco was used as a negative control, and changes in P9-1-GFP protein expression levels were detected using a Flag-specific antibody to evaluate the protein degradation efficiency of each E3 ligand.

[0135] Figure 4 and Figure 5 The results showed that, 4 days after injection, the expression level of P9-1-GFP did not change significantly in either the control group (P9-1-GFP alone or co-injected with OBP15 and OBP40). However, when P9-1-GFP was co-injected with OBP15-SPOP or OBP40-SPOP, the GFP fluorescence intensity showed a significant dose-dependent decrease (see...). Figure 4 c-4d, Figure 5 c-5d). Western blot analysis showed that the expression level of P9-1-GFP decreased significantly after injection of OBP15-SPOP and OBP40-SPOP, and the expression level of P9-1-GFP was almost undetectable when the concentration of OBP40-SPOP was 0.1%. This indicates that both OBP15-SPOP and OBP40-SPOP can mediate the targeted degradation of P9-1-GFP, and OBP40-SPOP can efficiently mediate the targeted degradation of P9-1-GFP (see c-5d). Figure 4 b、 Figure 5 b). Further protein structure analysis revealed the interaction pattern between OBP40-SPOP and P9-1-GFP (see [link]). Figure 4 a, Figure 5 a).

[0136] Example 4: OBP15-SPOP and OBP40-SPOP specific degradation of P9-1 protein To verify whether the specific degradation of P9-1-GFP originates from the specific recognition and targeting of P9-1 protein by the protein design binders OBP15 and OBP40, GFP protein was used as the target protein. Using an Agrobacterium-mediated transient transformation method, vhhGFP4-SPOP, OBP15-SPOP, and OBP40-SPOP vectors were co-transformed with the target protein vector into *Nicotiana benthamiana* leaves, with vhhGFP4 co-transformed with a reporter vector as a control. Finally, Western blotting analysis was used to evaluate the degradation efficiency of GFP protein, thereby clarifying the specific targeting effect of OBP15 and OBP40 on P9-1 protein.

[0137] Figure 6 The results showed that 4 days after injection, there was no significant change in GFP expression levels in either the control group (GFP alone or co-injected with vhhGFP4). However, compared to the control group, GFP expression levels were significantly reduced after co-injection with vhhGFP4-SPOP. Figure 6 The results showed that co-injection of GFP with OBP15-SPOP or OBP40-SPOP did not significantly change the expression level of GFP. This indicates that vhhGFP4-SPOP can specifically recognize and degrade GFP protein through the vhhGFP4 ligand, while OBP15-SPOP and OBP40-SPOP cannot recognize GFP protein through the OBP15 and OBP40 ligands. This suggests that the P9-1-GFP specific degradation mediated by OBP15-SPOP and OBP40-SPOP depends on the recognition and targeting of P9-1 protein by OBP15 and OBP40, rather than on the GFP protein itself.

[0138] Example 5: OBP15-SPOP and OBP40-SPOP confer RBSDV resistance in wheat. To verify whether bioPROTAC molecules (OBP15-SPOP and OBP40-SPOP) can specifically target and degrade the natural P9-1 protein in wheat, interfere with the assembly process of RBSDV, and achieve the goal of crop resistance to RBSDV.

[0139] As described in Section 4 of "Materials and Methods," the infective cDNA cloning rescue system of BYSMV-Nicotiana benthamiana-Planthopper-Monocotyledonous Crops was used to express bioPROTAC molecules (OBP15-SPOP and OBP40-SPOP) between the N and P proteins of the BYSMV-EGFP-RFP vector, with BYSMV-EGFP-RFP (BY-GR), BYSMV-EGFP-OBP15 (BY-OBP15), and BYSMV-EGFP-OBP40 (BY-OBP40) serving as controls. Subsequently, using the BYSMV reverse genetics platform, an equal volume mixture of Agrobacterium carrying the above recombinant vector, pcb301-NPL, and pGD-VSRs was injected into tobacco leaves. Approximately 13 days later, significant GFP fluorescence expression was observed in the tobacco leaves. After extracting tobacco juice, it was injected into planthoppers to allow the virus to replicate within them. The infected planthoppers were then transferred to wheat seedlings, ensuring approximately 3-4 planthoppers per seedling. After about a week, the infected planthoppers were blown out, and the wheat was cultured until obvious fluorescence was observed in the wheat leaves (see...). Figure 7 a) After the bioPROTAC molecule was expressed in large quantities in wheat, wheat was then infected with planthoppers carrying RBSDV. Based on the wheat leaf phenotype, immunoblotting analysis of the RBSDV capsid protein P10 in infected wheat leaves, and quantitative analysis of the RBSDV capsid protein CP gene in infected wheat leaves, the resistance of wheat to RBSDV designed with the novel bioPROTAC molecule was jointly evaluated.

[0140] Figure 7 The results showed that the bioPROTAC molecules OBP15-SPOP and OBP40-SPOP were normally expressed in wheat leaves. Figure 7 The results showed that in the control group, wheat leaves expressing BY-GR, BY-OBP15, and BY-OBP40 exhibited obvious yellow-white streaks after RBSDV infection. In wheat leaves expressing BY-OBP15-SPOP, the yellow-white streaks were lessened; and in wheat leaves expressing BY-OBP40-SPOP, the reduction in symptoms was more significant. This indicates that the expression of BY-OBP15-SPOP and BY-OBP40-SPOP can enhance wheat resistance to RBSDV to some extent, with BY-OBP40-SPOP showing a more pronounced antiviral effect.

[0141] To further verify the degradation efficiency of natural P9-1 protein and resistance to RBSDV by OBP15-SPOP and OBP40-SPOP in wheat, RNA was extracted from wheat leaves infected with RBSDV, and the expression level of the RBSDV coat gene CP was analyzed by qRT-PCR. Figure 7 The results showed that, compared with wild-type (WT), wheat leaves expressing BY-GR, BY-OBP15, and BY-OBP40 had significantly lower CP content. This phenomenon may be attributed to the fact that after BYSMV infection, intracellular resources available for viral replication in wheat cells are preferentially utilized by BYSMV, thereby limiting the replication capacity of subsequently infected RBSDV, leading to a decrease in RBSDV replication levels in wheat leaves. Further analysis revealed that, compared with BY-OBP15, wheat leaves expressing BY-OBP15-SPOP had lower CP content; while wheat leaves expressing BY-OBP40-SPOP had significantly lower CP content.

[0142] Figure 7 Western blot analysis of proteins showed that, compared with wild-type (WT), the content of P10 protein in RBSDV was significantly reduced in wheat leaves expressing BY-OBP15 and BY-OBP40, a result consistent with CP quantitative analysis. Furthermore, the expression level of P10 protein was further decreased in wheat leaves expressing BY-OBP15-SPOP and BY-OBP40-SPOP.

[0143] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims of the present invention.

Claims

1. Applications of protein degradation-targeting chimeras in plant antiviral therapy or plant breeding.

2. The use according to claim 1, characterized in that, The protein degradation targeting chimera includes a target protein binding domain and a target protein degradation domain; Preferably, the target protein binding domain comprises one or more of monomers, antibodies, antibody fragments, scaffold proteins, peptide conjugates, or ligands; more preferably, the target protein binding domain comprises a domain that binds to plant virus proteins. Preferably, the target protein degradation domain includes ubiquitin ligase and / or ubiquitin-binding enzyme; Preferably, the protein degradation targeting chimera is a bioPROTAC molecule; Preferably, the target protein binding domain and the target protein degradation domain are directly linked or linked via a linker; more preferably, the linker is a polypeptide linker; more preferably, the linker has an amino acid sequence as shown in general formula (G n S) m or an amino acid sequence having 1, 2, or 3 insertions, substitutions, or deletions of amino acids compared to the amino acid sequence as shown in general formula (G n S) m (G), n, m are each an integer from 1 to 10.

3. The use according to claim 1 or 2, characterized in that, The plants include crops, wild plants, and artificially cultivated non-crop plants; Preferably, the crop includes one or more of food crops, cash crops, vegetable crops, fruit crops, or forage crops; and / or The plant target proteins include plant virus proteins; Preferably, the plant target protein includes one or more of the following: capsid protein, envelope protein, nucleocapsid protein, replication protein, motility protein, RNA silencing repressor, or pathogenic protein of plant viruses.

4. A polypeptide that specifically binds to the P9-1 protein of rice black-streaked dwarf virus, comprising any one of the amino acid sequences shown in SEQ ID NO: 3-6, or a conserved variant of any one of the amino acid sequences shown in SEQ ID NO: 3-6 obtained by adding, deleting, substituting or modifying one or more amino acids.

5. A bioPROTAC molecule comprising a target protein binding domain and a target protein degradation domain, wherein the target protein binding domain comprises the polypeptide of claim 4.

6. The bioPROTAC molecule according to claim 5, characterized in that, The target protein degradation domain includes ubiquitin ligases and / or ubiquitin-binding enzymes. Preferably, the ubiquitin ligase includes one or more of SPOP, HECT, and RING; Preferably, the ubiquitin-binding enzyme includes Ube2A, Ube2B, Ube2D1, UBE2D2, UBE2D3, Ube2D4, or mutants thereof; Preferably, the target protein binding domain and the target protein degradation domain are directly linked or linked through a linker; more preferably, the linker is a peptide linker; even more preferably, the linker has a general formula (G n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m The amino acid sequences shown are compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, where n and m are integers from 1 to 10.

7. An isolated nucleic acid molecule encoding the polypeptide of claim 4 or the bioPROTAC molecule of claim 5 or 6.

8. A recombinant expression vector comprising the nucleic acid molecule of claim 7; Preferably, the recombinant expression vector includes a plant expression vector, and more preferably a plant binary expression vector.

9. A host cell containing the nucleic acid molecule of claim 7 or the recombinant expression vector of claim 8, or expressing the polypeptide of claim 4 or the bioPROTAC molecule of claim 5 or 6.

10. The use of the polypeptide of claim 4, the bioPROTAC molecule of claim 5 or 6, the nucleic acid molecule of claim 7, the recombinant expression vector of claim 8, or the host cell of claim 9 in plant antiviral or plant breeding; Preferably, the plant is a grass (Poaceae). More preferably, the plant includes one or more of rice, corn, wheat, barley, sorghum, and millet; Preferably, the plant target protein includes plant virus protein; Preferably, the plant target protein includes one or more of the following: capsid protein, envelope protein, nucleocapsid protein, replication protein, motility protein, RNA silencing repressor, and pathogenic protein of plant viruses; preferably, it is the P9-1 protein of rice black-streaked dwarf virus. Preferably, the plant antiviral activity includes plant resistance to rice black-streaked dwarf virus.

11. A method for ubiquitinizing and degrading plant target proteins or regulating the expression level of plant target proteins, comprising the step of contacting a protein degradation targeting chimera with the plant target protein. Preferably, the plant target protein includes plant virus protein; More preferably, the plant target protein includes one or more of the following: capsid protein, envelope protein, nucleocapsid protein, replication protein, motility protein, RNA silencing repressor, and pathogenic protein of plant viruses, preferably the P9-1 protein of rice black-streaked dwarf virus.

12. A method for plant antiviral therapy, comprising the step of causing a protein degradation-targeting chimera to be expressed in the plant.

13. The method according to claim 11 or 12, characterized in that, The protein degradation targeting chimera includes a target protein binding domain and a target protein degradation domain; Preferably, the target protein binding domain comprises one or more of monomers, antibodies, antibody fragments, scaffold proteins, peptide conjugates, or ligands; more preferably, the target protein binding domain comprises a domain that binds to plant virus proteins. Preferably, the target protein degradation domain includes ubiquitin ligase and / or ubiquitin-binding enzyme; Preferably, the protein degradation targeting chimera is a bioPROTAC molecule; Preferably, the binding domain and the degradation domain are directly connected or connected via a linker; more preferably, the linker is a peptide linker; even more preferably, the linker has a general formula (G n S) m The amino acid sequence shown, or the sequence with general formula (G n S) m The amino acid sequences shown are compared to amino acid sequences with 1, 2, or 3 inserted, substituted, or deleted amino acids, where n and m are integers from 1 to 10. Preferably, the protein degradation targeting chimera comprises the bioPROTAC molecule as described in claim 5 or 6; Preferably, the plants include crops, wild plants, and artificially cultivated non-crop plants; more preferably, the crops include one or more of food crops, cash crops, vegetable crops, fruit crops, and forage crops. Preferably, the plant is a grass; more preferably, the plant includes one or more of rice, corn, wheat, barley, sorghum, and millet.

14. The method according to any one of claims 11-13, characterized in that, The method includes the step of introducing a nucleic acid molecule containing an encoding a protein degradation-targeting chimera into the plant; Preferably, the method includes a transformation step using an Agrobacterium-mediated transformation method; More preferably, the method includes the following steps: S1: Transform a viral vector containing a nucleic acid molecule encoding the protein degradation targeting chimera into a viral expression host; S2: The transformed virus expression host is transformed into the plant using a virus-specific vector; Preferably, step S1 includes: transforming the viral vector into Agrobacterium tumefaciens, and then injecting the transformed Agrobacterium tumefaciens into a dicotyledonous plant; preferably, the dicotyledonous plant is a leaf of Nicotiana benthamiana. Preferably, step S2 includes: transforming a virus extracted from the dicotyledonous plant into a planthopper, and then infecting the plant with the planthopper, thereby causing the plant to express the protein degradation targeting chimera.

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