Plant endogenous small molecule polypeptide and application thereof in inhibiting RNA virus infected plants

Abstract

The invention discloses a plant endogenous small molecule polypeptide and application thereof in inhibiting RNA virus infected plants, the sequence of the small molecule polypeptide is Pep1-3: IIGVSNFDPVKRS, and the small molecule polypeptide is derived from nicotiana benthamiana; the invention relates to application of plant endogenous small molecule polypeptide in inhibiting RNA virus infection of plants. The plant is tobacco; the RNA viruses comprise tobacco mosaic viruses, tomato mosaic viruses and turnip mosaic viruses, and the small peptide Pep1-3 serves as a novel plant immunity inducer and shows the potential of the small peptide Pep1-3 in the aspect of preventing and controlling the RNA viruses. In agricultural production, prevention and treatment of virus diseases by adopting oligopeptide substances are a green and sustainable direction, and compared with traditional pesticides and plant immunity inducers, the composition has the advantages of broad spectrum, small toxicity, no drug resistance risk and long lasting period, and can generate a synergistic or synergistic effect when being mixed with other medicaments.

Description

Technical Field

[0001] This invention relates to the field of plant endogenous small molecule polypeptide technology, specifically to a plant endogenous small molecule polypeptide and its application in inhibiting RNA virus infection in plants. Background Technology

[0002] Plant viral diseases, also known as "plant cancer," are the second leading cause of plant disease outbreaks. Because viruses lack independent metabolic capabilities and rely on the host cell's metabolic network for replication, assembly, and release, plant infection can cause severe damage. Viral diseases account for approximately one-third of all plant diseases in globally important economic crops annually. Currently, control of plant viral diseases primarily relies on chemical control. However, there are currently no effective chemical pesticides to control viral diseases, and their application is limited due to increasing viral resistance and the environmental and human health impacts of excessive pesticide use. Utilizing resistant varieties is currently the most environmentally friendly and efficient method for controlling viral diseases. However, plant viruses mutate rapidly, leading to the loss of varietal resistance. Furthermore, the selection and breeding of resistant varieties, whether through conventional breeding methods or genetic engineering, is time-consuming and cannot be quickly applied to disease control. Therefore, developing new methods for controlling viral diseases is crucial. Summary of the Invention

[0003] The purpose of this invention is to provide a plant endogenous small molecule polypeptide and its application in inhibiting RNA virus infection in plants, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a plant endogenous small molecule polypeptide, wherein the sequence of the small molecule polypeptide is Pep1-3: IIGVSNFDPVKRS, derived from Nicotiana benthamiana.

[0005] Application of plant endogenous small molecule peptides in inhibiting RNA virus infection in plants.

[0006] Preferably, the plant is tobacco.

[0007] Preferably, the RNA virus includes tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and turnip mosaic virus (TuMV).

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: Pep1-3, as a novel plant immune inducer, has demonstrated its potential in controlling RNA viruses; in agricultural production, the use of short peptides for the prevention and control of viral diseases will be a green and sustainable direction. Compared with traditional pesticides, plant immune inducers have a broad spectrum, low toxicity, no risk of drug resistance, long duration of action, and can produce synergistic or enhanced effects when used in combination with other agents. Attached Figure Description

[0009] Figure 1 To illustrate the present invention, this study observed fluorescent images of different viruses (TMV, ToMV, and TuMV) in plants under UV lamp irradiation with and without Pep1-3 treatment. Detailed Implementation

[0010] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] Please see Figure 1 The present invention provides a technical solution: a plant endogenous small molecule polypeptide, wherein the sequence of the small molecule polypeptide is Pep1-3: IIGVSNFDPVKRS, derived from Nicotiana benthamiana.

[0012] The application of plant endogenous small molecule peptides in inhibiting RNA virus infection, wherein the plant is tobacco, and the RNA viruses include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and turnip mosaic virus (TuMV).

[0013] Example:

[0014] 1. Short peptide processing and inoculation

[0015] 1) Based on the short peptide sequence, the short peptide was synthesized at a biotechnology company (Shanghai Sangon Biotech) and diluted with sterile water to a final concentration of 300 μg / mL.

[0016] Two weeks of growth of *Nicotiana benthamiana* were selected and sprayed evenly on the leaves. Sterile water was used as a control group. The amount of water used in the treatment group and the control group was kept consistent throughout the process.

[0017] 2) After treatment with short peptides for 6 hours, TMV, ToMV, and TuMV were inoculated using Agrobacterium tethering (all three viruses were tagged with GFP, and the viral infection could be observed under a UV lamp).

[0018] The virus inoculation method is as follows:

[0019] Agrobacterium carrying infectious clones of TMV-GFP, ToMV-GFP, and TuMV-GFP were inoculated into 2 mL of YEP liquid medium and cultured at 28 °C with shaking for 12–16 hours. The cells were then collected by centrifugation at 5000 rpm for 2 min.

[0020] The bacterial cells were suspended in an infection solution (containing 10 mM MES, 10 mM MgCl2, and 200 μM As), and the bacterial concentration was adjusted to OD600 = 0.0001. 0.2 mL of the obtained Agrobacterium suspension was injected into leaves (inoculated leaves) using a sterile syringe. After injection, the injected leaves were divided into two groups, each containing three biological replicates.

[0021] The injection area was approximately 1 square centimeter, located within the small peptide treatment area and at the same position on the leaf. The inoculated *Nicotiana benthamiana* was cultured at 25°C, and symptoms were observed. Western blotting was used to analyze the accumulation of virus in the inoculated leaves and systemic leaves. The observation period for TMV, ToMV, and TuMV was 5-7 days.

[0022] Configure the Mock (control group) and Pepl-3 (experimental group) settings:

[0023] Mock (Control Group): The control group received no additional exogenous proteins. In this section, prominent green fluorescence images are visible, indicating a high viral load and a significant increase in viral protein expression levels. This high viral protein expression level suggests that viral infection and replication are normal in the absence of exogenous intervention, without any additional attenuation or inhibition.

[0024] Pepl-3 (Experimental Group): This group included the addition of PepL-3 protein. In this section, the green fluorescence image is noticeably weaker, indicating lower viral protein expression levels. This suggests that viral expression was significantly inhibited by exogenous treatment. This indicates that the PepL-3 protein has an inhibitory effect on certain aspects of the virus, affecting viral expression and replication within the plant.

[0025] During the observation of the experiment, it was found that:

[0026] TMV (Tobacco Mosaic Virus): In fluorescence microscopy images, TMV exhibits green fluorescence, indicating the distribution and concentration of the virus within cells.

[0027] The results showed that the protein expression level of TMV was significantly reduced in the PepL-3 experimental group, indicating that the PepL-3 protein has a certain inhibitory effect on TMV infection or replication.

[0028] ToMV (Tomato Mosaic Virus): ToMV appears as a green fluorescence, and the accumulation and distribution of the virus in plant cells are also observed in this way.

[0029] Experimental results showed that PepL-3 protein also inhibited the protein expression level of ToMV, exhibiting a significant reduction.

[0030] TuMV (Turnip Mosaic Virus): TuMV appears as a green fluorescence, and the fluorescent image clearly shows the distribution of the virus.

[0031] The addition of PepL-3 protein also significantly reduced the protein expression level of TuMV, confirming its effect in this virus.

[0032] Protein expression analysis: The expression levels of viral proteins were detected by methods such as Western blotting.

[0033] Mock group: When PepL-3 protein was not added, the protein expression levels of each virus were high.

[0034] Pepl-3 group: After the addition of PepL-3 protein, the protein expression levels of each virus were significantly reduced, especially the protein expression levels of TMV, ToMV and TuMV were significantly reduced.

[0035] Experimental results showed that, under UV light, the infection of TMV, ToMV, and TuMV viruses was observed. Compared with the control group, the accumulation of viruses in the inoculated leaves and systemic leaves of Nicotiana benthamiana treated with small peptide Pep1-3 was significantly lower. This indicates that small peptide Pep1-3 is not conducive to virus infection. Pep1-3 can be used as a green and safe immune inducer. Small peptide Pep1-3 has good potential for agricultural application and can be used for the prevention and control of plant viral diseases.

[0036] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A plant endogenous small molecule polypeptide, characterized in that: The sequence of the small molecule polypeptide is Pep1-3: IIGVSNFDPVKRS, derived from Nicotiana benthamiana.

2. The application of the plant endogenous small molecule polypeptide of claim 1 in inhibiting RNA virus infection of plants.

3. The application of a plant endogenous small molecule polypeptide according to claim 2 in inhibiting RNA virus infection in plants, characterized in that: The plant in question is tobacco.

4. The application of a plant endogenous small molecule polypeptide according to claim 3 in inhibiting RNA virus infection in plants, characterized in that: The RNA viruses include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and turnip mosaic virus (TuMV).

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