Use of compound 35c3 in inducing plant immunity and enhancing plant disease resistance

Abstract

This invention discloses the application of compound 35C3 in inducing plant immunity and enhancing plant disease resistance, belonging to the field of biopesticide technology. Experiments have shown that compound 35C3 can effectively activate the phosphorylation of plant mitogen-activated protein kinase, induce reactive oxygen species (ROS) bursts, and upregulate the expression levels of disease resistance-related genes FRK1, PR1, PR2, WRKY29, and WRKY53. Pathogen inoculation experiments demonstrated that 35C3 can significantly enhance the resistance of Arabidopsis thaliana to *Pseudomonas syringae* and significantly enhance the resistance of poplar to *Colletotrichum gloeosporioides* and *Botrytis cinerea*. Its mechanism of action is to activate the plant's own immune system rather than directly inhibiting pathogen growth. Furthermore, 35C3 elicits immune responses in various crops, showing broad-spectrum applicability. This invention provides a new approach for developing environmentally friendly biopesticides and has significant agricultural application value.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to the application of compound 35C3 in inducing plant immunity and enhancing plant disease resistance. Background Technology

[0002] Through long-term co-evolution with pathogens and herbivorous insects, plants have evolved two immune systems: pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Pathogen-associated molecular patterns (such as bacterial flagella and fungal chitin) are ubiquitous and relatively conserved in microorganisms and can be specifically recognized by pattern recognition receptors on the surface of plant cells, thereby triggering the PTI response. This process effectively defends against the invasion of most pathogenic microorganisms by closing stomata, unleashing reactive oxygen species, activating the mitogen-activated protein kinase (MAPK) cascade, and synthesizing antimicrobial substances. When pathogens successfully invade plant cells, the toxic effector proteins they secrete can inhibit PTI signaling. At this time, the NB-LRR receptor protein in the plant can directly or indirectly recognize these effector proteins, inducing a stronger ETI response, manifested as increased levels of hormone signaling molecules, a surge in reactive oxygen species, cell wall lignification, and even leading to hypersensitive reactions (HR) and programmed cell death.

[0003] Plant immune inducers (also known as plant vaccines) are a class of biological pesticides that enhance plant resistance to diseases and pests by activating the plant's own immune system. Unlike traditional pesticides that act directly on pathogens or pests, immune inducers induce broad-spectrum and durable resistance in plants by inducing the expression of resistance genes, regulating metabolic pathways, promoting the accumulation of resistance substances, and stimulating systemic resistance. Based on their origin, plant immune inducers can be divided into natural small-molecule and synthetic small-molecule inducers.

[0004] Diseases are a key factor restricting agricultural production. To control diseases, some regions have long relied on chemical pesticides, which has not only led to the gradual accumulation of pesticide resistance in pathogens and pests, but also caused a series of environmental problems such as soil degradation and water pollution, damaging the stability of agricultural ecosystems. Furthermore, excessive pesticide residues can be transmitted through the food chain, posing a potential threat to human health. Therefore, improving the plant's own resistance while reducing the use of chemical pesticides has become an urgent need for green agricultural development. Plant immune inducers, with their environmental friendliness, broad spectrum of action, and low likelihood of inducing resistance, have become a research hotspot in the field of plant protection.

[0005] The small molecule compound 35C3 is an organic acid containing a peptide bond, and its structural formula has been published in the literature. Current research on this compound mainly focuses on chemical synthesis, while its use as a plant immune inducer to activate plant immunity and enhance disease resistance has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to provide a synthetically produced plant immune inducer and its application. Specifically, this invention discovers that the small molecule compound 35C3 can effectively activate the phosphorylation of mitogen-activated protein kinases (MAPKs) and induce the expression of a series of related disease resistance genes. It can also significantly enhance the plant's resistance to pathogens such as *Pseudomonas syringae* (Pst DC3000), *Colletotrichum gloeosporioides*, and *Botrytis cinerea*.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Application of compound 35C3 in the preparation of products for inducing plant immunity and / or enhancing plant disease resistance.

[0009] In some embodiments, the enhancement of plant disease resistance includes enhancing the plant's resistance to bacterial and / or fungal diseases.

[0010] In some embodiments, the bacterial disease is caused by *Pseudomonas syringae*.

[0011] In some embodiments, the fungal disease is caused by Colletotrichum gloeosporioides or Botrytis cinerea.

[0012] In some embodiments, the plant includes at least one of Arabidopsis thaliana, poplar, tomato, mung bean, green bean, soybean, eggplant, pumpkin, corn, wheat, tulip tree, ginkgo and sunflower.

[0013] In some embodiments, the concentration of compound 35C3 used in the product is 1-100 μM.

[0014] In some embodiments, the concentration of compound 35C3 used in the product is 5-20 μM.

[0015] In some embodiments, the concentration of compound 35C3 used in the product is 10 μM.

[0016] A method for inducing plant immunity and enhancing plant disease resistance includes the step of treating plants with an effective dose of compound 35C3.

[0017] A plant immune inducer whose active ingredient includes compound 35C3.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) This invention reveals for the first time the potential of the small molecule compound 35C3 as a plant immune activator, opening up entirely new application areas for known compounds. This provides new candidate molecules and ideas for the creation of green pesticides.

[0020] (2) Through rigorous experiments, this invention has demonstrated that 35C3 does not act directly on pathogens, but rather enhances the plant's immune level by activating the plant's own MAPK signaling pathway, promoting reactive oxygen species release and the expression of disease-resistant genes. Its immune activation effect is significant on various model plants and crops.

[0021] (3) 35C3 can effectively enhance the resistance of plants to different types of pathogens (including bacteria and fungi), and has broad-spectrum control potential, which is superior to traditional single-target chemical pesticides.

[0022] (4) As a plant immune inducer, 35C3 resists diseases by enhancing the plant's own immunity. It does not directly kill pathogens, so it is not easy to develop drug resistance. It also has low toxicity to humans and animals, is environmentally friendly, and meets the requirements of sustainable agricultural development. Attached Figure Description

[0023] Figure 1 Diagram showing the activation of FRK1 expression in plants by treatment with the small molecule compound 35C3;

[0024] Figure 2 The diagram shows the immune response in Arabidopsis thaliana induced by the immune inducer 35C3 used in this embodiment of the invention. A: ROS accumulation in Arabidopsis thaliana leaves detected by DAB staining; B: Relative DAB staining intensity per unit leaf area in Figure A; C: ROS burst in Arabidopsis thaliana seedlings after 35C3 treatment determined by L012 chemiluminescence method; D: Increased phosphorylation level of Arabidopsis thaliana MAPK protein after 35C3 treatment; EI: Increased expression level of disease resistance-related genes in Arabidopsis thaliana after 35C3 treatment.

[0025] Figure 3 The diagram shows the immune response in poplar trees induced by the immune inducer 35C3 used in this embodiment of the invention. A: ROS accumulation in poplar leaves detected by DAB staining; B: Relative DAB staining intensity per unit leaf area in Figure A; C: ROS burst in poplar trees after 35C3 treatment determined by L012 chemiluminescence method; D: Increased phosphorylation level of poplar MAPK protein after 35C3 treatment; EI: Increased expression level of disease resistance-related genes in poplar trees after 35C3 treatment.

[0026] Figure 4 This diagram illustrates the hypersensitivity reactions induced in various crops by the immune inducer 35C3 used in the embodiments of this invention.

[0027] Figure 5 Figure 1 shows the enhancement of Arabidopsis thaliana resistance to Pseudomonas syringae strain Pst. DC3000 by the immune inducer 35C3 used in this embodiment of the invention. A: Different concentrations of 35C3 treatment had no significant effect on the growth of Pst. DC3000. B: The area of ​​Pst. DC3000 colonies in Figure A. C: Photographs of Pst. DC3000 colonies growing on plates on Arabidopsis thaliana leaves after Mock (1% DMSO) treatment and 10 μM 35C3 treatment. D: Colony density statistics in Figure C.

[0028] Figure 6 The following diagram illustrates how the immune inducer 35C3 used in this invention enhanced the resistance of poplar to anthracnose. A: Different concentrations of 35C3 had no significant effect on the growth of the anthracnose pathogen in poplar. B: Colony area of ​​*Colletotrichum gloeosporioides* in diagram A. C: Pretreatment of '84K' poplar leaves with 10 μM 35C3 significantly improved the resistance of poplar to *Colletotrichum gloeosporioides*. D: Statistical results of the area of ​​diseased parts in diagram C.

[0029] Figure 7 The following diagram illustrates how the immune inducer 35C3 used in this invention enhanced the resistance of poplar to gray mold. A: Different concentrations of 35C3 had no significant effect on the growth of the gray mold pathogen in poplar. B: The colony area of ​​*Botrytis cinerea* in Figure A. C: Pretreatment of '84K' poplar leaves with 10 μM 35C3 significantly improved the resistance of poplar to gray mold. D: Statistical results of the area of ​​diseased parts in Figure C. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] As mentioned in the background section, although plants possess an immune system, its function is somewhat delayed, requiring time to respond to pathogen invasion. This negatively impacts disease mitigation / prevention or control. Furthermore, plants have limited immunity levels, thus necessitating the use of plant immune inducers to accumulate resistance. In previous research, the inventors of this application discovered that the small molecule compound 35C3 can promote the generation and accumulation of reactive oxygen species in plants, increase the phosphorylation level of MAPK proteins, and enhance the expression levels of disease resistance genes such as FRK1, PR1, PR2, WRKY29, and WRKY53. The chemical formula of the small molecule compound 35C3 is shown below:

[0032]

[0033] To further develop new plant immune inducers, this invention conducted experiments on the bioactivity of the small molecule compound 35C3. The results showed that 35C3 can stimulate the immune response of plants and enhance their resistance to fungal and bacterial diseases. Applying it before the peak of disease outbreaks can prevent diseases from occurring in the first place.

[0034] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0035] Unless otherwise specified in the embodiments of this invention, all consumables are common laboratory consumables in the art and can be obtained through ordinary commercial channels.

[0036] The plant immune inducer used in the embodiments of the present invention is prepared according to the following method:

[0037] A 10 mM 35C3 stock solution was prepared using DMSO as the solvent, and then diluted with sterile water to a working concentration of 10 μM. All control groups in the experiment were prepared with DMSO diluted to the same concentration and proportion.

[0038] Example 1:

[0039] 1. Experimental plants: The pGWB535 recombinant expression vector with the GUS gene driven by the FRK1 promoter was constructed, transformed into Agrobacterium GV3101 strain, and then transformed into wild-type Arabidopsis thaliana. The Arabidopsis pFRK1-GUS transgenic lines obtained after screening and identification were used as experimental plants.

[0040] 2. Planting method:

[0041] To test the immunomodulatory activity of the screened small molecule compound 35C3, different concentrations of the compound were applied to the pFRK1-GUS transgenic line. The Arabidopsis seeds of the pFRK1-GUS transgenic line were washed with sterile water (30% commercial bleach and 0.02% Triton X-100) for 16 min, then washed more than 5 times with sterile water in a laminar flow hood, and placed in a 4 ℃ refrigerator for 1-2 days. Solid culture medium containing 2.25 g / L MS, 5 g / L sucrose, and 0.4% plant gel was melted by heating and added to each well of a 96-well plate using a pipette. The sterilized seeds were resuspended in 0.1% agarose and spotted into 96-well clear plates, and cultured at 23 ℃ under 16 h light / 8 h dark conditions for 5-6 days.

[0042] 3. Experimental methods:

[0043] Add different concentration gradients of small molecule 35C3 and its analogues (preferably enough to cover the seedlings in the wells) dissolved in 100 μL of ddH2O to each well of a 96-well plate. The specific concentrations are 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM. A bacterial flagellar peptide Flg22 at a final concentration of 100 nM was used as a positive control for 5 h. After removing the small molecule solutions, 100 μL of GUS staining solution (100 mM NaH2PO4, 10 mM Na2EDTA, 0.5 mM K4[Fe(CN)6]·3H2O, 0.5 mM K3[Fe(CN)6], 0.1% Triton X-100, 0.4 mg / mL X-Gluc, adjusted to pH 7.0) was added, and staining was performed at 37 ℃ for 4-5 h. After removing the staining solution, the plate was destained with 95% ethanol, and the staining was observed.

[0044] 4. Experimental Results:

[0045] The results are as follows Figure 1 As shown, different concentrations of 35C3 treatment can induce the production of blue substances in Arabidopsis thaliana, indicating that 35C3 promotes the expression of the FRK1 gene in Arabidopsis thaliana.

[0046] Example 2:

[0047] 1. Experimental plants: Arabidopsis thaliana (Col-0) and Populus alba × P. glandulosa, '84K'.

[0048] 2. Planting method:

[0049] Seedlings used for immune activation experiments need to be disinfected with disinfectant (30% 84 disinfectant + 70% sterile water + 0.1% Triton-X100) and placed at 4°C for two days. Then, they are sown on 1 / 2 MS medium or nutrient soil and placed in a greenhouse for cultivation. The greenhouse conditions are: 16 h of light, 8 h of darkness, and constant temperature of 24°C.

[0050] 3. Experimental methods:

[0051] For the DAB staining experiment, rosette leaves of Arabidopsis thaliana that have grown for four weeks or '84K' leaves that have been transplanted for four weeks were treated with different concentrations of 35C3 and then vacuum-permeated with 1 mg / mL DAB staining solution (containing 0.05% Tween 20). After incubation overnight in the dark, the leaves were decolorized by boiling with ethanol-glacial acetic acid (3:1) until the chlorophyll was completely removed, and the reddish-brown ROS precipitate was observed.

[0052] For the reactive oxygen species (ROS) burst experiment, 5-6 day old Arabidopsis seedlings or '84K' leaf discs transplanted four weeks prior were immersed in ddH2O, with a single seedling placed in each well. The wells were then covered and incubated overnight at room temperature. 50 μL of a reaction mixture containing the specified concentration of the test compound, 40 μM L-012, and 20 μg / mL horseradish peroxidase (HRP) was added to each well. This reaction mixture was then rapidly injected into each well. The luminescence values ​​were immediately measured using a LUX-P110 microplate luminescence detector.

[0053] For the assay to detect the phosphorylation level of MAPK protein, Arabidopsis seedlings treated with 10 μM 35C3 were homogenized to extract crude protein, which was then immediately extracted in protein extraction buffer. The resulting homogenate was centrifuged at 4°C for 10 minutes. The supernatant was collected for SDS-PAGE. Immunoblot analysis of phosphorylated MAPKs was performed using primary antibody (p44 / 42MPKs (1:2000)) and secondary antibody (peroxidase-conjugated goat anti-rabbit IgG (1:15000)).

[0054] For the analysis of disease resistance gene expression levels, total RNA was extracted from Arabidopsis thaliana and poplar using the Kangwei Century Ultrapure RNA kit or RNApureFast Plant Kit, reverse transcribed using the Kangwei Century HiFiScript SuperFast gDNARemoval cDNA Synthesis Kit, and real-time fluorescence quantitative analysis was performed using the Kangwei Century SuperStar Blue UniversalSYBR Master Mix kit.

[0055] Experimental results:

[0056] The results are as follows Figure 2 and Figure 3 As shown, 35C3 treatment can promote the release and accumulation of ROS in 5-6 day old Arabidopsis seedlings, 4-week-old Arabidopsis leaves, and 4-week-old tissue culture seedlings of Populus '84', and can significantly increase the phosphorylation level of MAPK protein and the expression level of disease resistance genes.

[0057] Example 3:

[0058] 1. Experimental plants: Arabidopsis thaliana (Col-0), tomato, mung bean, green bean, soybean, eggplant, pumpkin, corn, wheat, tulip tree, ginkgo and sunflower.

[0059] 2. Experimental Methods:

[0060] Healthy leaves of common herbaceous and woody plants were injected with 35C3 at concentrations of 10 μM, 20 μM, 50 μM, and 100 μM, and the changes in the leaves were observed.

[0061] Experimental results:

[0062] The results are as follows Figure 4 As shown, 35C3 can induce hypersensitivity reactions in a variety of plants, causing rapid necrosis of cells at the inoculation site, forming localized dry lesions, which worsen with increasing 35C3 concentration.

[0063] Example 4:

[0064] 1. Experimental plant: Arabidopsis thaliana (Col-0)

[0065] 2. Planting method:

[0066] Seedlings used for the Pseudomonas syringae (Pst DC3000) infection experiment need to be disinfected with disinfectant (30% 84 disinfectant + 70% sterile water + 0.1% Triton-X100) and placed at 4℃ for two days. Then, they are sown on nutrient soil and placed in a greenhouse for cultivation. The greenhouse conditions are: 16 hours of light, 8 hours of darkness, and a constant temperature of 24℃.

[0067] 3. Experimental methods:

[0068] Add 10 μL of Pst DC3000 (OD) 600 0.6) Inoculate on solid culture medium containing different concentrations of 35C3 (10 μM, 20 μM, 50 μM), and observe and count the size of the plaques after 2 days.

[0069] Select 4-5 week old Arabidopsis leaves of uniform size. Prepare 2 mL each of mock (1% DMSO) and 10 μM 35C3. Inject the leaves with a 1 mL syringe and label them. Return the injected plants to the greenhouse and cover them to maintain humidity after 30 min. 48 h after compound injection, inoculate with Pst DC3000. 48 h after inoculation, remove the leaves and disinfect them with 75% alcohol for 30 s in a clean bench. Puncture the center of the inoculated Arabidopsis leaves to obtain samples. The removed leaves were placed in 1.5 mL centrifuge tubes containing steel balls. 500 μL of 10 mM sterile magnesium chloride solution (MgCl2) was added to the centrifuge tubes. The tubes were then placed in a centrifuge and shaken at 40 Hz for 30 seconds, followed by uniform shaking on a vertical shaker for half an hour to obtain a pre-prepared bacterial culture. This pre-prepared bacterial culture was then diluted 100,000 times. The diluted culture was then plated on 9 cm diameter transparent plates using King's B (KB, Hangzhou Microbial Co., Ltd.) solid medium supplemented with rifampicin (0.05 mg / mL, filtered and sterilized). The plates were incubated in the dark at 28°C. After 2-3 days, the number of colonies on the medium was counted. The calculation method was density = number of colonies per plate. Dilution factor / disc area (cfu / cm²) 2 Statistics on the growth status of Pst DC3000.

[0070] Experimental results:

[0071] The results are as follows Figure 5 As shown, 35C3 does not directly inhibit the growth of Pst DC3000; however, pretreatment of Arabidopsis thaliana with 10 μM 35C3 for 48 h can significantly enhance the resistance of Arabidopsis thaliana to Pst DC3000.

[0072] Example 5

[0073] 1. Experimental plant: Silver glandular poplar (Populus alba × P. glandulosa, '84K')

[0074] 2. Planting method:

[0075] Agar blocks (3 mm in diameter) containing fresh hyphae of *Colletotrichum gloeosporioides* (Cg) and *Botrytis cinerea* (Bc) were inoculated onto a solid culture medium containing a specified concentration of 35C3. The growth of the pathogens was observed, and photographs were taken and the data collected after 2 days of incubation.

[0076] The '84K' poplar seedlings used for pathogen infection were removed from the tissue culture bottles and cultured in a greenhouse under the following conditions: 16 hours of light, 8 hours of darkness, and a constant temperature of 24 ℃. Four weeks after transplanting, poplar leaves of uniform size were selected, soaked in 75% ethanol for 30-40 seconds, thoroughly rinsed with sterile water, and finally blotted with sterile filter paper to remove residual moisture. A 10 μM 35C3 working solution was prepared, and 10 μL of the compound solution was added to designated areas of the leaves. The leaves were then placed in a vacuum dryer and continuously evacuated for 5 minutes to promote compound penetration. The leaves were then cultured in a tissue culture room for 48 hours. 48 hours after inoculation, 10 μL of spore suspension (Colletotrichum gloeosporioides and Botrytis cinerea) was inoculated, and the leaves were continuously evacuated for 5 minutes. The leaves were then cultured in a tissue culture room.

[0077] Experimental results:

[0078] The results are as follows Figure 6 and Figure 7 As shown, 35C3 does not directly inhibit the growth of *Colletotrichum gloeosporioides* and *Botrytis cinerea*, however, pretreatment with 10 μM 35C3 can significantly enhance the resistance of '84K' poplar to *Colletotrichum gloeosporioides* and *Botrytis cinerea*.

[0079] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. Application of compound 35C3 in the preparation of products for inducing plant immunity and enhancing plant disease resistance.

2. Use according to claim 1, characterized in that, The enhancement of plant disease resistance includes enhancing plant resistance to bacterial and fungal diseases.

3. The application according to claim 2, characterized in that, The bacterial disease is caused by *Pseudomonas syringae*.

4. The application according to claim 2, characterized in that, The fungal disease is caused by Colletotrichum gloeosporioides or Botrytis cinerea.

5. The application according to any one of claims 1-4, characterized in that, The plants include at least one of Arabidopsis thaliana, poplar, tomato, mung bean, green bean, soybean, eggplant, pumpkin, corn, wheat, tulip tree, ginkgo and sunflower.

6. The application according to claim 5, characterized in that, The concentration of compound 35C3 used in the product is 1-100 μM.

7. The application according to claim 6, characterized in that, The concentration of compound 35C3 used in the product is 5-20 μM.

8. The application according to claim 7, characterized in that, The concentration of compound 35C3 used in the product is 10 μM.

9. A method for inducing plant immunity and / or enhancing plant disease resistance, characterized in that, This includes the step of treating plants with an effective dose of compound 35C3.

10. A plant immune inducer, characterized in that, Its active ingredient includes compound 35C3.

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