Use of VdGH7-6 protein to increase plant resistance and / or to induce an immune response in plants

Abstract

The application provides application of VdGH7-6 protein in improving plant resistance and / or inducing plant immune response, a method for improving plant resistance and / or inducing plant immune response, and application of the VdGH7-6 protein in preparation of pesticides. The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO. 1. The VdGH7-6 protein can be used to efficiently induce plant ROS burst, callose deposition, expression of resistance-related genes and other immune responses, and has important significance in agricultural production.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, and more specifically, to the application of VdGH7-6 protein in enhancing plant resistance and / or 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] Current research on the pathogenic mechanism of Verticillium dahliae has identified a variety of CWDEs-like proteins. Due to the diversity of the interaction between CWDEs proteins and the host, their efficiency in inducing plant immune responses varies. Therefore, it is of great significance to discover host immune-activating proteins that better meet production needs. 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 VdGH7-6 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 VdGH7-6 protein in the preparation of pesticides.

[0005] To achieve the above objectives, the first aspect of this disclosure provides the use of VdGH7-6 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 Staphylococcus aureus and Verticillium dahliae;

[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, Staphylococcus aureus and Verticillium dahliae.

[0008] Optionally, the concentration of the VdGH7-6 protein used is 20-40 μM, with the optimal concentration being 25 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 VdGH7-6 protein is applied to the target plant, and / or the gene encoding the VdGH7-6 protein is introduced into the target plant.

[0012] The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1;

[0013] The nucleotide sequence of the gene encoding the VdGH7-6 protein is shown in SEQ ID NO.2.

[0014] Optionally, the gene encoding the VdGH7-6 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 Staphylococcus aureus and Verticillium dahliae;

[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 Staphylococcus aureus and Verticillium dahliae.

[0018] On the other hand, this disclosure provides the use of VdGH7-6 protein in the preparation of pesticides, optionally, the pesticides are used to improve plant resistance and / or induce the immune response of plants, the amino acid sequence of the VdGH7-6 protein being shown in SEQ ID NO.1.

[0019] Optionally, the plant includes tobacco.

[0020] Through the above technical solution, this disclosure provides that the VdGH7-6 protein can act to stimulate plant immune responses, and can efficiently induce plant ROS burst, callus deposition, and expression of resistance-related genes at low concentrations, while significantly enhancing plant resistance and immunity, 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 VdGH7-6 protein in Example 1 of this disclosure.

[0023] Figure 2 This is a diagram showing the area of ​​lesions on the leaves of plants treated with VdGH7-6 protein and GFP in Example 2 of this disclosure.

[0024] Figure 3 This is a diagram showing the experimental results of VdGH7-6 protein inducing plant resistance gene expression in Example 2 of this disclosure.

[0025] Figure 4 This is a diagram showing the experimental results of VdGH7-6 protein inducing the expression of disease resistance-related genes in plants in Example 3 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 VdGH7-6 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 VdGH7-6 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, VdGH7-6 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 Staphylococcus aureus and Verticillium dahliae;

[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, Staphylococcus aureus and Verticillium dahliae.

[0031] According to this disclosure, the concentration of the VdGH7-6 protein used is 20-40 μM, preferably 25 μM. The VdGH7-6 protein provided by this disclosure can significantly improve plant resistance and induce plant immune responses at relatively low concentrations.

[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 VdGH7-6 protein is applied to the target plant, and / or the gene encoding the VdGH7-6 protein is introduced into the target plant.

[0035] The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1;

[0036] The nucleotide sequence of the gene encoding the VdGH7-6 protein is shown in SEQ ID NO.2.

[0037] According to this disclosure, the gene encoding the VdGH7-6 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 Staphylococcus aureus and Verticillium dahliae;

[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] This disclosure also provides the application of VdGH7-6 protein in the preparation of pesticides for enhancing plant resistance and / or inducing an immune response in plants, wherein the amino acid sequence of VdGH7-6 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 VdGH7-6 protein disclosed in this embodiment has the amino acid sequence shown in SEQ ID NO.1. The preparation method of the VdGH7-6 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] MKTNFGVLLAFLAAARAQQACTSQAETHPPLTWQKCTGTGGSSCTNVSGSVVLDSNWRWTHTVSGSTN

[0047] CYDGNKWTASCGTDGTTCAQQCCVDGADYANTYGVTTSGNALTLKFVTKHQYGTNIGSRVYLMENDT

[0048] KYQAFNLLGNEFTFDVDVSKTGCGLNGALYFVSMDLDGGVSKYSTNKAGAKYGTGYCDSQCPRDVKFI

[0049] GGVANVAGWVPAADDPNSGVGNLGACCAEMDIWEANKISTAYTPHPCQNNAYHTCQGDKCGGTYSA

[0050] DRYGGDCDPDGCDFNSYRQGDKTFYGEGTGFKLNTAQKLTVVTQFLRSGGSTGDLSEIKRFYVQNGVV

[0051] FANSQSTVAGNPGNSLTPAFCKAQKTAFGDQNIFDQRGGFAQTSDAVSKPMVLVMSLWNDHYANMLW

[0052] LDSTFPTTSTGPGAERGSCPTTSGTPADTETNQANATVQFSNIKFGPIGSTFNQGAVSPPSGGTTVVAPPPA

[0053] TTLATSTRAAVTTTTRAAPPTTTTASSGGAAAPKWGQCGGQGWTGPTTCVAGSTCQAQNQWYSQCL

[0054] SEQ ID NO.2:

[0055]

[0056] Example 1

[0057] VdGH7-6 protein stimulates plant immune response:

[0058] (1) VdGH7-6 protein induces ROS outbreak in plants

[0059] Four-week-old tobacco plants were selected, and approximately 20 μL of 25 μM glycoside hydrolase VdGH7-6 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. 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 stain, destained with 95% ethanol, and observed under a stereomicroscope.

[0060] Results: Compared with the 1×PBS control, the tobacco leaves injected with the VdGH7-6 protein region showed significant DAB staining, indicating a large accumulation of reactive oxygen species. Figure 1 As shown.

[0061] The above experiments show that glycoside hydrolase VdGH7-6 can induce ROS bursts in tobacco leaves.

[0062] Example 2

[0063] VdGH7-6 induces a resistance response in tobacco to Botrytis cinerea:

[0064] Using a 1 mL syringe without a needle, inject approximately 20 μL of 25 nM glycoside hydrolase VdGH7-6 protein into the abaxial surface of 4-week-old tobacco leaves. Use 1×PBS (pH 7.5) as a negative control. 12-24 hours after injection, puncture the injection area with a needle and inject 5 μL of pre-prepared 5×10... 6 A drop of spores / mL was placed directly above the needle puncture site. The lesions were incubated at 25°C and 80% humidity. After 3-5 days of inoculation, the diameter of the lesions in each treatment was observed and measured, and biomass was assessed. Figure 2-3 As shown, the area of ​​lesions on the experimental leaves was approximately 210 mm². 2 The area of ​​lesions on the control leaf was approximately 813 mm². 2 .

[0065] The above results indicate that VdGH7-6 protein can induce a resistance response in tobacco to Botrytis cinerea, and the lesion area is reduced by about 74.17%, which significantly improves the plant's resistance to the pathogen.

[0066] Example 3

[0067] Transient conversion of VdGH7-6 protein induces expression of resistance-related genes in tobacco leaves:

[0068] Four-week-old tobacco plants were selected, and the concentration of Agrobacterium pGR107-VdGH7-6 transiently transformed with VdGH7-6 was adjusted to OD. 600 0.5-0.8 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. 48 h 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.

[0069] Results: Compared with the control, the transcription level of disease resistance-related genes in the pGR107-VdGH7-6 region of tobacco leaves injected with the drug was significantly increased, such as... Figure 4 As shown.

[0070] The above experiments show that the glycoside hydrolase VdGH7-6 induces the expression of tobacco resistance genes.

[0071] It is evident that applying VdGH7-6 protein to plants can induce a strong plant immune response in a short period of time, with a strong plant immune response occurring in the protein injection area within 6 hours, including reactive oxygen species bursts and callose deposition; a strong plant immune response can be rapidly induced even at relatively low working concentrations.

[0072] 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.

[0073] 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.

[0074] 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 VdGH7-6 protein in enhancing plant resistance and / or inducing plant immune responses, characterized in that, The amino acid sequence of the VdGH7-6 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 Botrytis cinerea and Verticillium dahliae; The induced immune response in the plant is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, Botrytis cinerea and Verticillium dahliae. The plant in question is tobacco.

2. The application according to claim 1, wherein, The concentration of the VdGH7-6 protein used is 20-40 μM.

3. The application according to claim 2, wherein, The concentration of the VdGH7-6 protein used was 25 μM.

4. A method for enhancing plant resistance and / or inducing plant immune responses, characterized in that, The method includes the following steps: Apply the VdGH7-6 protein to the target plant, and / or introduce the gene encoding the VdGH7-6 protein into the target plant; The amino acid sequence of the VdGH7-6 protein is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the VdGH7-6 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 Botrytis cinerea and Verticillium dahliae; The induced immune response in the plant is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, Botrytis cinerea and Verticillium dahliae. The plant in question is tobacco.

5. The method according to claim 4, wherein, The gene encoding the VdGH7-6 protein was introduced into the target plant via a plant expression vector.

6. The method according to claim 4, wherein, The plant expression vector includes PGR107.

7. The application of VdGH7-6 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 VdGH7-6 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 Botrytis cinerea and Verticillium dahliae; The induced immune response in the plant is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, Botrytis cinerea and Verticillium dahliae. The plant in question is tobacco.

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