Small peptide taLEP1 and its use in plant antiviral defense

Abstract

The application discloses a small peptide TaLEP1 and application thereof in plant antiviral defense, and belongs to the field of agricultural biotechnology. An amino acid sequence of the plant antiviral defense small peptide TaLEP1 is shown as SEQ ID NO:1, and the small peptide TaLEP1 can be used for preparing plant antiviral products, including virus inhibitors and antiviral drugs, and is suitable for inhibiting wheat yellow mosaic virus, turnip mosaic virus and soybean mosaic virus of the Potyviridae family. When used, the TaLEP1 is dissolved in sterile PBS to prepare a 5 muM working solution, and is externally sprayed on plant leaves; after absorption of the plant, the TaLEP1 can induce the activation of a plant autophagy related pathway, and then inhibit virus infection and reduce the replication and accumulation of the virus in the plant. The small peptide TaLEP1 provided by the application can effectively inhibit the replication of various RNA viruses of the Potyviridae family, and has good antiviral effect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biology, and in particular relates to the small peptide TaLEP1 and its application in plant antiviral defense. Background Technology

[0002] Plant viral diseases are one of the major factors causing yield reductions in major food crops worldwide, among which the Potato Virus Y family (Potatovirus Y) Potyviridae Plant RNA viruses (PRNAVs) are the largest family of plant RNA viruses and are a significant type of virus that threatens crops and economic crops worldwide. Current research on plant antiviral mechanisms mainly focuses on regulatory aspects such as RNA silencing, immune receptor recognition, and plant hormone signaling. In recent years, the autophagy pathway has also been discovered to play a crucial role in clearing viral replication components.

[0003] Autophagy is a highly conserved degradation mechanism in eukaryotes, involving the formation of autophagosomes, a double-membrane structure, to encapsulate cellular components and transport them to vacuoles for degradation. In recent years, autophagy has been shown to play a crucial role in plant immunity, becoming an important component of antiviral defense. Plants can utilize selective autophagy to degrade key viral-encoded proteins. For example, NBR1 (NEIGHBOR OF BRCA1), as an autophagy receptor, can recognize and mediate the autophagic degradation of viral proteins such as the capsid protein of cauliflower mosaic virus (CaMV) and the RNA silencing repressor HC-Pro of turnip mosaic virus (TuMV).

[0004] Although previous studies have shown that the autophagy pathway plays an important role in plant antiviral defense and can inhibit viral infection to some extent, its regulatory mechanism remains unclear. Currently, there is a lack of natural molecules that can effectively activate plant autophagy and thus enhance the overall antiviral capacity of plants. In particular, in the field of plant endogenous small peptides, there are no systematic research reports on their induction of autophagy and inhibition of viral infection.

[0005] In addition, existing antiviral methods mostly rely on genetic modification or chemical treatment, which have problems such as limited application scope and high environmental risks. There is an urgent need to develop an antiviral active factor that is naturally derived, safe, and can be applied by exogenous spraying to achieve green control of plant viral diseases. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes the small peptide TaLEP1 and its application in plant antiviral defense. The small peptide TaLEP1 can effectively inhibit the replication of various RNA viruses of the Potato Virus Y family, exhibiting good antiviral effects.

[0007] To achieve the above objectives, the present invention provides a method derived from wheat ( Triticum aestivumThe small peptide TaLEP1 for plant antiviral defense, wherein the amino acid sequence of the small peptide TaLEP1 is MGDSVQERARRWLEVSYCSLSINPIFMYLCVFGTVLDLL, as shown in SEQ ID NO:1.

[0008] This invention provides the application of the above-mentioned small peptide TaLEP1 in the preparation of plant antiviral defense products, wherein the virus is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus of the Potato Virus Y family.

[0009] Preferably, the product includes a virus inhibitor or an antiviral drug.

[0010] Preferably, the present invention also provides a plant antiviral defense inhibitor, wherein the plant antiviral defense inhibitor comprises the above-mentioned small peptide TaLEP1.

[0011] Preferably, the inhibitor is a water-based solvent, emulsion, slow-release colloid, or foliar spray formulation.

[0012] This invention provides the application of the above-mentioned small peptide TaLEP1 or the above-mentioned plant antiviral defense inhibitor in inhibiting viral infection, wherein the virus is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus of the Potato Virus Y family.

[0013] This invention provides the application of the above-mentioned small peptide TaLEP1 or the above-mentioned plant antiviral defense inhibitor in improving the antiviral defense ability of plants, wherein the virus is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus of the Potato Virus Y family.

[0014] Preferably, the present invention also provides a method for inhibiting plant virus infection using the small peptide TaLEP1, comprising the following steps:

[0015] (1) Dissolve the above small peptide TaLEP1 in sterile PBS to prepare a 5 μM working solution;

[0016] (2) Apply exogenous spray to plant leaves to enable them to absorb the small peptide TaLEP1;

[0017] (3) After spraying, TaLEP1 induces the activation of plant autophagy-related pathways, thereby inhibiting plant virus infection.

[0018] This invention provides a method for enhancing plant antiviral ability using the small peptide TaLEP1. Plants are treated with the aforementioned small peptide TaLEP1 to induce autophagy, thereby enhancing the plant's antiviral ability.

[0019] Preferably, the TaLEP1 can be obtained by chemical synthesis and has a purity >90%.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects:

[0021] This invention proposes a natural small peptide, TaLEP1, and its effect on improving plant resistance to Potato Virus Y (Potatovirus Y). Potyviridae The application of TaLEP1 in the resistance of plant viruses provides a new approach and feasibility for enhancing plant resistance using non-classical small peptides. Resistance identification experiments against wheat yellow mosaic virus (WYMV), turnip mosaic virus (TuMV), and soybean mosaic virus (SMV) showed that exogenous spraying of TaLEP1 significantly reduced viral infection rates and viral RNA accumulation, inhibited viral spread within plants, and demonstrated a broad-spectrum antiviral effect. Molecular-level analysis revealed that TaLEP1 promotes the formation of ATG8-labeled autophagosomes, significantly enhancing the plant's autophagy defense response, thereby effectively inhibiting viral infection by activating the autophagy pathway.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 SDS-PAGE electrophoresis image of purified TaLEP1 (Coomassie brilliant blue staining).

[0024] Figure 2 This is a Western blot image of TaLEP1 after purification;

[0025] Figure 3 The image shows the viral symptoms after TaLEP1 spraying. In the image, A represents wheat yellow mosaic virus (WYMV), B represents turnip mosaic virus (TuMV), and C represents soybean mosaic virus (SMV).

[0026] Figure 4 The images show the quantitative accumulation map of viral RNA and the fluorescence spot area map. Among them, A is the accumulation map of wheat yellow mosaic virus (WYMV), B is the quantitative fluorescence spot area map of turnip mosaic virus (TuMV), and C is the accumulation map of soybean mosaic virus (SMV).

[0027] Figure 5 The viral protein accumulation was detected by WB, where A is wheat yellow mosaic virus (WYMV), B is turnip mosaic virus (TuMV), and C is soybean mosaic virus (SMV).

[0028] Figure 6 A confocal plot showing the number of autophagosomes. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0031] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0032] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0033] The expression vector pET-32a(+) is commercially available.

[0034] Example 1

[0035] TaLEP1 expression and purification:

[0036] The preparation of the TaLEP1 small peptide described in this invention was carried out by Huaan Biotechnology Co., Ltd., using a prokaryotic expression system for in vitro expression and purification.

[0037] First, based on the amino acid sequence of the small peptide TaLEP1 (SEQ ID NO:1: MGDSVQERARRWLEVSYCSLSINPIFMYLCVFGTVLDLL), its codons were optimized to suit E. coli. Escherichia coli The expression preference of [a specific gene] was determined, and the coding sequence was cloned into the expression vector pET-32a(+) to construct the recombinant plasmid pET32a-TaLEP1. A His tag was fused to the N-terminus of this vector to facilitate subsequent affinity purification.

[0038] Transform the recombinant plasmid into E In .coli BL21(DE3) competent cells, positive clones were selected and induced with IPTG (final concentration 0.5mM) and cultured in a shaker at 16℃ for 12h to promote soluble expression.

[0039] After induction, the bacterial cells were collected by centrifugation and sonicated with lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM Midazole, pH 8.0) to obtain the supernatant.

[0040] supernatant Purification was performed using NTA affinity chromatography (Ni-Sepharose 6FF, Cytiva), with the target protein eluted using an elution gradient containing 50 mM to 250 mM Imidazole.

[0041] After dialysis to remove imidazole, the elution product was analyzed by SDS-PAGE, showing a single characteristic band of approximately 25 kDa. The results are as follows: Figure 1 As shown in the figure. Western blot analysis using an anti-LEP1 monoclonal antibody confirmed that the expressed protein was the TaLEP1 fusion protein, as shown in the figure. Figure 2 As shown. The purified product was lyophilized and stored at -80°C for later use.

[0042] Example 2

[0043] Application of TaLEP1 exogenous spraying to inhibit viral infection:

[0044] The lyophilized and purified TaLEP1 was dissolved in sterile PBS to prepare a 5 μM working solution; the control group used an equal volume of PBS.

[0045] Take healthy wheat and tobacco ( N.benthamiana The TaLEP1 solution was sprayed onto both sides of the leaves until moist, approximately 5 mL per plant. After treatment, the plants were placed in a greenhouse (28℃ / 25℃, 15h light / 9h darkness) for 24h.

[0046] Wheat was inoculated with wheat yellow mosaic virus (WYMV) infection solution after treatment; tobacco was inoculated with turnip mosaic virus TuMV-GFP; and soybean was inoculated with soybean mosaic virus (SMV). Both WYMV and SMV viruses were inoculated using the mechanical friction method, maintaining humidity above 70% after infection. TuMV virus was inoculated with *Agrobacterium tumefaciens* using the Agrobacterium tumefaciens infiltration method on *Nicotiana benthamiana* (…). Nicotiana benthamiana Inoculate the plants with the virus. Observe the phenotype and virus accumulation at 7 and 10 days post-infection. The antiviral phenotype of the treated plants is as follows: Figure 3 As shown.

[0047] The results showed that, compared with the PBS control group, the TaLEP1 sprayed group showed significantly reduced symptoms 7 days after inoculation: the yellow streaks on the leaves of wheat yellow mosaic virus (WYMV) infected plants were significantly reduced (e.g., Figure 3 (A); Tobacco ( N.benthamiana The fluorescence signal of TuMV-GFP was significantly weakened and its distribution range was reduced (e.g., Figure 3 (B) After soybeans were inoculated with soybean mosaic virus (SMV), leaf curling, necrosis, and chlorosis were significantly reduced (e.g., Figure 3(C). Viral RNA levels were detected using qRT-PCR. After inoculation, the viral RNA accumulation in the plants was detected by qRT-PCR. Figure 4 (As shown).

[0048] The results showed that the viral RNA content in the TaLEP1 treatment group was significantly lower than that in the PBS control group, with WYMV decreasing by approximately 72%, TuMV-GFP by approximately 65%, and SMV by approximately 80%, all with statistically significant differences (P<0.01). Western blot analysis of viral protein accumulation using antiviral coat protein antibodies yielded the following results: Figure 5 As shown in the figure, compared with the control group, the accumulation of capsid protein (CP) of the three viruses in the TaLEP1 treatment group was significantly reduced, and the gray value of the band decreased by more than 70% compared with the control, indicating that viral protein synthesis was significantly inhibited.

[0049] The results in summary indicate that exogenous application of TaLEP1 peptide can effectively inhibit the replication and accumulation of various plant viruses in different hosts.

[0050] Example 3

[0051] TaLEP1 induces autophagy:

[0052] To verify whether TaLEP1 enhances plant antiviral capabilities by inducing host autophagy, this example uses tobacco ( Nicotiana benthamiana Using GFP-ATG8f as a model, the autophagy marker protein was used for fluorescence localization observation and to detect the level of viral infection. An expression vector carrying the GFP-ATG8f fusion protein was constructed as a fluorescent indicator of autophagosome formation. TaLEP1-3×HA or an empty vector (control) was transiently transfected into... N.benthamiana Leaves. GFP fluorescence signal was observed using a laser confocal microscope 48 hours later.

[0053] The results are as follows Figure 6 As shown, in control leaves, the GFP-ATG8f signal was mainly diffusely distributed; while in leaves expressing TaLEP1, a large number of punctate GFP signals were observed, indicating a significant increase in the number of autophagosomes. This result indicates that TaLEP1 can induce the activation of the host autophagy pathway.

[0054] In summary, this invention clarifies the core characteristics and mechanism of action of the small peptide TaLEP1. The amino acid sequence of this small peptide is shown in SEQ ID NO:1, and it can be efficiently expressed and purified using a prokaryotic expression system (such as Escherichia coli BL21(DE3)). Its core antiviral mechanism lies in inducing the activation of plant autophagy-related pathways—by promoting the formation of ATG8-labeled autophagosomes, it enhances the plant's selective degradation ability of viral components, thereby inhibiting viral infection and replication at the molecular level, filling the research gap in the regulation of autophagy by endogenous small peptides in plant antiviral activity. Furthermore, this small peptide can be directly used to prepare plant antiviral products, including virus inhibitors and antiviral drugs, with applicable formulations covering water-soluble, emulsion, slow-release colloid, and foliar spray formulations (meeting the needs of convenient application in agricultural production). Its antiviral spectrum clearly targets key pathogenic viruses of the Potato Virus Y family, specifically including Wheat Yellow Mosaic Virus (WYMV), Turnip Mosaic Virus (TuMV), and Soybean Mosaic Virus (SMV), making it suitable for virus control in various crops such as wheat, soybean, and tobacco. At the same time, it has the advantages of green prevention and control. Compared with traditional genetic modification (limited application scope) and chemical agents (high environmental risk), TaLEP1 is naturally derived, has high safety, and can be effective by external spraying. It is simple to operate and easy to promote, providing a new technical path for the green prevention and control of plant viral diseases.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A plant antiviral defense peptide TaLEP1 derived from wheat, characterized in that, The amino acid sequence of the small peptide TaLEP1 is MGDSVQERARRWLEVSYCSLSINPIFMYLCVFGTVLDLL.

2. The application of the small peptide TaLEP1 according to claim 1 in the preparation of plant antiviral defense products, characterized in that, The virus in question is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus, belonging to the Potato Virus Y family.

3. The application according to claim 2, characterized in that, The products include virus inhibitors or antiviral drugs.

4. A plant antiviral defense inhibitor, characterized in that, The plant antiviral defense inhibitor includes the plant antiviral defense peptide TaLEP1 as described in claim 1.

5. The plant antiviral defense inhibitor according to claim 4, characterized in that, The inhibitor is an aqueous solvent, emulsion, or sustained-release colloid.

6. The application of the plant antiviral defense peptide TaLEP1 according to claim 1 or the plant antiviral defense inhibitor according to claim 4 in inhibiting viral infection, characterized in that, The virus in question is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus, belonging to the Potato Virus Y family.

7. The application of the plant antiviral defense peptide TaLEP1 according to claim 1 or the inhibitor according to claim 4 in improving plant antiviral defense capabilities, characterized in that, The virus in question is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus, belonging to the Potato Virus Y family.

8. A method for inhibiting plant virus infection using the plant antiviral defense peptide TaLEP1, characterized in that, The plant virus is wheat yellow mosaic virus and / or turnip mosaic virus and / or soybean mosaic virus belonging to the Potato Virus Y family. The method includes the following steps: 1) Dissolve the plant antiviral defense peptide TaLEP1 described in claim 1 in sterile PBS to prepare a working solution; 2) Apply exogenous spray to plant leaves to allow them to absorb the plant's antiviral defense peptide TaLEP1; 3) After spraying, TaLEP1 induces the activation of autophagy-related pathways in plants, thereby inhibiting plant virus infection.

9. A method for enhancing plant antiviral ability using the plant antiviral defense peptide TaLEP1, characterized in that, Plants treated with the plant antiviral defense TaLEP1 according to claim 1 induce autophagy, thereby enhancing the plant's resistance to wheat yellow mosaic virus, turnip mosaic virus, and soybean mosaic virus.

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