Application of VdPL9-3 protein in enhancing plant resistance and inducing plant immune responses

Abstract

This invention provides the application of VdPL9-3 protein in improving plant resistance and / or inducing plant immune responses. The amino acid sequence of VdPL9-3 protein is shown in SEQ ID NO.1. It is a highly efficient plant immune activating protein that can effectively improve plant resistance and enhance the host's ability to defend against pathogens.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, specifically relating to the application of VdPL9-3 protein in enhancing plant resistance and inducing plant immune responses. Background Technology

[0002] To overcome the plant cell wall defense barrier, pathogens secrete a series of cell wall degradative enzymes (CWDEs) during infection, aiding in their invasion and nutrient acquisition. However, CWDEs or plant molecules themselves can also act as activators, triggering plant immune responses such as stomatal closure, cell wall callose deposition, reactive oxygen species (ROS) bursts, and intracytoplasmic calcium release. 2+ Changes in concentration, cellular ion leakage, expression of disease-related genes, and synthesis of phytoalexins are all factors involved. Among the numerous plant defense responses induced by pathogens, ROS (Reactive Oxygen Species) play a crucial role and are an important means for plants to resist pathogen infection. Therefore, studying pathogenic CWDEs can provide a new perspective for the discovery of plant resistance genes and the breeding of disease-resistant plants.

[0003] Studies on the pathogenic mechanism of *Verticillium dahliae*, both domestically and internationally, have identified various CWDE-like proteins. Due to the diversity of CWDE protein interactions with the host, their efficiency in inducing plant immune responses varies. Therefore, discovering host immune-activating proteins that better meet production needs is of great significance. Summary of the Invention

[0004] To efficiently induce immune responses such as ROS outbreak, callus deposition, and expression of resistance-related genes in plants, this disclosure provides the application of VdPL9-3 protein in enhancing plant resistance and / or inducing plant immune responses, a method for enhancing plant resistance and / or inducing plant immune responses, and the application of VdPL9-3 protein in the preparation of pesticides.

[0005] To achieve the above objectives, the first aspect of this disclosure provides the use of VdPL9-3 protein in enhancing plant resistance and / or inducing plant immune responses, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus;

[0007] The induced immune response in the plant includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus.

[0008] Optionally, the concentration of the VdPL9-3 protein used is 40-100 μM, with the optimal concentration being 60-80 μM.

[0009] Optionally, the plant includes tobacco.

[0010] On the other hand, this disclosure provides a method for improving plant resistance and / or inducing plant immune responses, the method comprising the following steps:

[0011] The VdPL9-3 protein is applied to the target plant, and / or the gene encoding the VdPL9-3 protein is introduced into the target plant.

[0012] The amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the gene encoding the VdPL9-3 protein is shown in SEQ ID NO.2.

[0014] Optionally, the gene encoding the VdPL9-3 protein is introduced into the target plant via a plant expression vector.

[0015] Optionally, the plant expression vector includes pGR107.

[0016] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus;

[0017] Optionally, the induced immune response in the plant includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus.

[0018] Optionally, the pesticide is used to enhance plant resistance and / or induce an immune response in plants, and the amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1.

[0019] Optionally, the plant includes tobacco.

[0020] Through the above technical solution, this disclosure provides that the VdPL9-3 protein acts on the activation and induction of plant immune responses, which can efficiently induce plant ROS burst, callus deposition, expression of resistance-related genes and other immune responses, while significantly enhancing plant resistance, providing a new approach to improving plant resistance.

[0021] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0022] Figure 1 This is a diagram showing the experimental results of the ROS response in plant leaves induced by the VdPL9-3 protein in Example 1 of this disclosure.

[0023] Figure 2This is a diagram showing the experimental results of VdPL9-3 protein inducing callose accumulation in plant leaves in Example 1 of this disclosure.

[0024] Figure 3 This is a diagram showing the experimental results of VdPL9-3 protein inducing plant resistance gene expression in Example 1 of this disclosure.

[0025] Figure 4 This is a diagram showing the experimental results of the VdPL9-3 protein inducing plant resistance to pathogens in Example 2 of this disclosure. Detailed Implementation

[0026] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0027] The first aspect of this disclosure provides the use of VdPL9-3 protein in enhancing plant resistance and / or inducing plant immune responses, the amino acid sequence of which is shown in SEQ ID NO.1.

[0028] The inventors of this disclosure, through extensive research, discovered that injecting VdPL9-3 protein into plant leaves, such as tobacco leaves, resulted in the detection of ROS production and callose accumulation in the leaves, and significantly upregulated the expression levels of some immune response-related genes in the injected plants. Therefore, effective VdPL9-3 protein can be used to enhance plant resistance and / or induce plant immune responses.

[0029] According to this disclosure, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus;

[0030] The induced immune response in the plant includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus.

[0031] According to this disclosure, the concentration of the VdPL9-3 protein used is 40-100 μM, preferably 60-80 μM.

[0032] According to this disclosure, the plant includes tobacco.

[0033] On the other hand, this disclosure provides a method for improving plant resistance and / or inducing plant immune responses, the method comprising the following steps:

[0034] The VdPL9-3 protein is applied to the target plant, and / or the gene encoding the VdPL9-3 protein is introduced into the target plant.

[0035] The amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1;

[0036] The nucleotide sequence of the gene encoding the VdPL9-3 protein is shown in SEQ ID NO.2.

[0037] According to this disclosure, the gene encoding the VdPL9-3 protein is introduced into the target plant via a plant expression vector.

[0038] According to this disclosure, the plant expression vector includes pGR107.

[0039] According to this disclosure, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus;

[0040] According to this disclosure, the induced immune response in plants includes inducing a reduction in the damage to plants caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus.

[0041] According to this disclosure, the pesticide is used to enhance plant resistance and / or induce an immune response in plants, and the amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1.

[0042] According to this disclosure, the plant includes tobacco.

[0043] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0044] The VdPL9-3 protein disclosed in this embodiment has the amino acid sequence shown in SEQ ID NO.1. The preparation method of the VdPL9-3 protein includes: constructing a recombinant expression vector, expressing it in prokaryotes using *E. coli*, the expression vector containing a maltose-binding protein (MBP) tag, and purifying it using an MBP TrapHP pre-packed column to obtain the target protein.

[0045] SEQ ID NO.1:

[0046] MKFFSILVAVAPAALVSAADFYVATTGSDTAAGSLATPFKSIQVAVNKAAAGDTIYLRGGTHKPTTNIQITKSGTAAKPYTVTAYNSENVIIDGEALTGTPAALNAALANKDRGIFHIEKVNYWKFYKLTFINGPYGVYVRDGTNHYFERITTHDNYETGFHMEGALSNNQVVYLNSYRNRDPRKNGESADGFALKQGSGTGNVLRGARLWENVDDGLDFWEFKDRVTDTTIAWGNGVNRWNFSPFAGDGNGFKLGGLPSGSTGNADHIVNNCIAFGNAAKFTDNKQTGTFLFTRNTAYNNGAVGFQTSAVKATFQNNIAARNSKTTAQSGQTSLKSATSTGNSWNSSPVWTDASFKSVDVSLVKGARQANGKIVASNFLLPASGGNIGATTNWQ

[0047] SEQ ID NO.2:

[0048]

[0049] Example 1

[0050] VdPL9-3 protein stimulates plant immune response:

[0051] (1) VdPL9-3 protein induces ROS outbreak in plants

[0052] Four-week-old tobacco plants were selected, and approximately 20 μL of 80 μM VdPL9-3 protein was injected into the abaxial surface of the leaves using a 1 mL syringe (needle removed). 1×PBS (pH 7.5) was used as a negative control. Six hours after injection, the leaves were rinsed with deionized water and incubated in 1×DAB staining solution at 25°C in the dark for 8 hours. The leaves were then rinsed with deionized water to remove residual staining, destained with 95% ethanol, and observed under a stereomicroscope. Results: Compared with the 1×PBS control, the areas of tobacco leaves injected with VdPL9-3 protein showed significantly more DAB staining, indicating a large accumulation of reactive oxygen species (ROS). Figure 1 As shown in the figure. The above experiments demonstrate that the VdPL9-3 protein can induce ROS bursts in tobacco leaves.

[0053] (2) VdPL9-3 protein induces callose accumulation in plants.

[0054] Four-week-old tobacco plants were selected, and approximately 20 μL of 80 μM VdPL9-3 protein was injected into the abaxial surface of the leaves using a 1 mL syringe (needle removed). 1×PBS (pH 7.5) solution was used as a negative control. Two hours after injection, the leaves were washed with deionized water, decolorized with 95% ethanol, and residual ethanol was removed with deionized water. The leaves were then placed in 150 mM phosphate buffer (pH 9.5) containing 0.1% aniline blue (w / v) and treated in the dark for 2 hours, followed by observation under a fluorescence microscope. Results: Compared with the 1×PBS control, the area of ​​tobacco leaves injected with VdPL9-3 protein showed a significant increase in the characteristic blue bright spots of callosity, such as... Figure 2 As shown in the figure. The above experiments indicate that VdPL9-3 protein can promote callose accumulation in tobacco leaves.

[0055] (3) VdPL9-3 protein induces the expression of genes related to plant immune response.

[0056] Four-week-old tobacco plants were selected, and the concentration of Agrobacterium pGR107-VdPL9-3 transiently transformed with VdPL9-3 was adjusted to OD. 6000.5-0.8 μL of Agrobacterium suspension was injected from the abaxial surface of the leaf using a 1 mL syringe (needle removed). Agrobacterium of the same concentration, transiently expressing pGR107-GFP, served as a negative control. Forty-eight hours after injection, leaves from the injected area were collected, total RNA was extracted, and quantitative PCR was used to detect the transcriptional levels of resistance-related genes. Results: Compared with the control, the transcriptional levels of disease resistance-related genes in the pGR107-VdPL9-3 region of tobacco leaves were significantly increased, such as... Figure 3 As shown in the figure. The above experiments demonstrate that the VdPL9-3 protein induces the expression of tobacco resistance genes.

[0057] Example 2

[0058] VdPL9-3 protein induces plant resistance to pathogens.

[0059] Four-week-old tobacco plants were selected, and approximately 20 μL of 80 μM VdPL9-3 protein was injected into the abaxial surface of the leaves using a 1 mL syringe (needle removed). 1×PBS (pH 7.5) was used as a negative control. Twelve hours after injection, 4-5 small holes were punctured in the injection area, and 5 μL of 5×10⁻⁶ PBS was dripped into each hole. 6 Staphylococcus griseus spore suspension per mL, after moisturizing for 3-5 days, observe and measure the diameter of lesions in each treatment, such as Figure 4 As shown.

[0060] The above experiments show that the VdPL9-3 protein activates the tobacco's defense response against Staphylococcus aureus, resulting in a reduction in lesion area.

[0061] In summary, VdPL9-3 protein can efficiently activate plant immune function, effectively improve plant resistance, enhance host pathogen defense capabilities, and significantly induce plant resistance responses to pathogens.

[0062] The preferred embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0063] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0064] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. The application of VdPL9-3 protein in enhancing plant resistance and / or inducing plant immune responses, characterized in that, The amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1; The improvement of plant resistance refers to improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus; The induced immune response in the plant is to reduce the harm to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus. The plant in question is tobacco.

2. The application according to claim 1, wherein, The concentration of the VdPL9-3 protein used is 40-100 μM.

3. The application according to claim 2, wherein, The concentration of the VdPL9-3 protein used is 60-80 μM.

4. A method for enhancing plant resistance and / or inducing plant immune responses, characterized in that, The method includes the following steps: Apply VdPL9-3 protein to the target plant, and / or introduce the gene encoding VdPL9-3 protein into the target plant; The amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the VdPL9-3 protein is shown in SEQ ID NO.2; The improvement of plant resistance refers to improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus; The induced immune response in the plant is to reduce the harm to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus. The plant in question is tobacco.

5. The method according to claim 4, wherein, The gene encoding the VdPL9-3 protein was introduced into the target plant via a plant expression vector.

6. The method according to claim 4, wherein, The plant expression vector was pGR107.

7. The application of VdPL9-3 protein in pesticide preparation, characterized in that, The pesticide is used to enhance plant resistance and / or induce an immune response in plants, and the amino acid sequence of the VdPL9-3 protein is shown in SEQ ID NO.1; The improvement of plant resistance refers to improving the plant's resistance to at least one pathogen among Verticillium dahliae and Staphylococcus aureus; The induced immune response in the plant is to reduce the harm to the plant caused by at least one of the pathogens, Verticillium dahliae and Staphylococcus aureus. The plant in question is tobacco.

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