Application of VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses

Abstract

This disclosure provides a method for the VdGH45-1 protein to activate and induce plant immune responses, which can efficiently induce immune responses such as ROS burst, callus deposition, and expression of resistance-related genes in plants, while significantly enhancing plant resistance, providing a new approach to improving plant resistance.

Description

Technical Field

[0001] This disclosure belongs to the field of microbial technology, specifically relating to the application of VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses. Background Technology

[0002] To successfully invade and multiply in host plants, pathogens secrete effectors between and within host cells, interfering with the host's normal physiological activities. It is generally believed that plant pathogenic fungal effectors exert their pathogenic function primarily through various means, including inducing reactive oxygen species (ROS) bursts, regulating plant gene transcription, stressing the host endoplasmic reticulum, manipulating plant immune secretion pathways, targeting host hormone signaling, interfering with autophagy, and manipulating host protein localization. When effectors invade plants, resistance genes can be activated by their corresponding effectors, participating in the resistance response. In modern resistance breeding, effectors have become important tools for resistance gene identification, functional analysis, and molecular breeding for disease resistance.

[0003] Researchers have identified more than 10 effectors that can stimulate plant immune responses in response to the interaction between Verticillium dahliae and its host. However, the efficiency of these effectors in stimulating plant immune responses varies, mainly in terms of the time required to stimulate the immune response and the protein concentration required.

[0004] Therefore, there is an urgent need to provide an effector that can induce immune responses such as ROS outbreaks and callose deposition in plants with high efficiency, significantly improve plant resistance and defense response, and provide a new approach for developing plant immune inducers and improving plant resistance. Summary of the Invention

[0005] 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 VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses, a method for enhancing plant resistance and / or inducing plant defense responses, and the application of VdGH45-1 protein in the preparation of pesticides.

[0006] To achieve the above objectives, the first aspect of this disclosure provides the use of VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Staphylococcus aureus and Verticillium dahliae;

[0008] The induced plant defense response includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Staphylococcus aureus and Verticillium dahliae.

[0009] Optionally, the concentration of the VdGH45-1 protein used is 2-10 μM, preferably 4-6 μM.

[0010] Optionally, the plant includes tobacco.

[0011] On the other hand, this disclosure provides a method for improving plant resistance and / or inducing plant defense responses, the method comprising the following steps:

[0012] The VdGH45-1 protein is applied to the target plant, and / or the gene encoding the VdGH45-1 protein is introduced into the target plant;

[0013] The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1;

[0014] The nucleotide sequence of the gene encoding the VdGH45-1 protein is shown in SEQ ID NO.2.

[0015] Optionally, the gene encoding the VdGH45-1 protein is introduced into the target plant via a plant expression vector.

[0016] Optionally, the plant expression vector includes pGR107.

[0017] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Staphylococcus aureus and Verticillium dahliae;

[0018] The induced plant defense response includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Staphylococcus aureus and Verticillium dahliae.

[0019] On the other hand, this disclosure provides the application of VdGH45-1 protein in the preparation of pesticides for improving plant resistance and / or inducing plant defense responses;

[0020] The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1.

[0021] Optionally, the plant includes tobacco.

[0022] Through the above technical solution, this disclosure provides that the VdGH45-1 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.

[0023] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0024] Figure 1 This shows the accumulation of reactive oxygen species in tobacco leaf areas after injection of VdGH45-1 protein and PBS.

[0025] Figure 2 This involves the determination of lesion area in tobacco leaves injected with VdGH45-1 and GFP proteins.

[0026] Figure 3 This study measured the pathogen biomass in tobacco leaf regions injected with VdGH45-1 and GFP proteins.

[0027] Figure 4 This study measured the transcriptional levels of disease resistance-related genes in leaf regions injected with pGR107-VdGH45-1 and pGR107-GFP negative controls. Detailed Implementation

[0028] 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.

[0029] The first aspect of this disclosure provides the use of VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses, the amino acid sequence of which is shown in SEQ ID NO.1.

[0030] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Staphylococcus aureus and Verticillium dahliae;

[0031] The induced plant defense response includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Staphylococcus aureus and Verticillium dahliae.

[0032] Optionally, the concentration of the VdGH45-1 protein used is 2-10 μM, preferably 4-6 μM.

[0033] Optionally, the plant includes tobacco.

[0034] On the other hand, this disclosure provides a method for improving plant resistance and / or inducing plant defense responses, the method comprising the following steps:

[0035] The VdGH45-1 protein is applied to the target plant, and / or the gene encoding the VdGH45-1 protein is introduced into the target plant;

[0036] The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1;

[0037] The nucleotide sequence of the gene encoding the VdGH45-1 protein is shown in SEQ ID NO.2.

[0038] Optionally, the gene encoding the VdGH45-1 protein is introduced into the target plant via a plant expression vector.

[0039] Optionally, the plant expression vector includes pGR107.

[0040] Optionally, the improvement of plant resistance includes: improving the plant's resistance to at least one pathogen among Staphylococcus aureus and Verticillium dahliae;

[0041] The induced plant defense response includes inducing a reduction in the damage to the plant caused by at least one of the pathogens, Staphylococcus aureus and Verticillium dahliae.

[0042] On the other hand, this disclosure provides the application of VdGH45-1 protein in the preparation of pesticides for improving plant resistance and / or inducing plant defense responses;

[0043] The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1.

[0044] Optionally, the plant includes tobacco.

[0045] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0046] The preparation method of VdGH45-1 protein includes: constructing a recombinant expression vector, expressing it in prokaryotes using Escherichia 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.

[0047] The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1:

[0048] MHANLLTHLLPLALHLTPTAAQASGSGSTTRYWDCCKPSCAWPGKAALSSGPVTTCDINDRPLGDANARSGCEAGGTSYMCSGQSPWAVSADLAYGWAAVRVAGGDEASWCCACYELT FTSGPVAGKKMVVQATNTGADLGSNHFDLAIPGGGVGIYNACTAQYNAPPNGWGDQYGGIRSRAECDAFPEALKSGCYWRFDWFLNADNPQVNFRQVSCPAALTSRSGCVRQDNAMFK

[0049] The nucleotide sequence encoding the VdGH45-1 protein is shown in SEQ ID NO.2:

[0050] ATGCACGCCAACCTCCTGACCCATCTCCTCCCCCTCGCCCTCCACCTGACGCCTACAGCAGCCCAGGCCTCCGGCAGCGGCAGCACAACCCGCTACTGGGACTGCTGCAAGCCCTCGTGCGCCTGGCCCGGCAAGGCGGCCCTCTCGAGCGGGCCGGTCACGACCTGCGACATCAACGACCGGCCGCTCGGCGACGCCAACGCGCGCTCGGGCTGCGAGGCCGGCGGCACGTCCTACATGTGCAGCGGGCAGTCGCCGTGGGCCGTGTCGGCGGACCTCGCGTACGGGTGGGCGGCCGTGCGCGTGGCGGGCGGCGACGAGGCGTCGTGGTGCTGCGCGTGCTACGAGCTTACGTTCACGAGCGGGCCGGTGGCTGGGAAGAAGATGGTTGTGCAGGCGACGAACACGGGGGCCGACCTCGGCTCGAACCACTTTGACCTTGCGATCCCTGGCGGCGGAGTCGGCATATACAACGCCTGCACGGCGCAGTACAACGCGCCGCCCAACGGCTGGGGCGACCAGTACGGCGGCATCCGCAGCCGCGCCGAGTGCGACGCCTTCCCGGAGGCGCTCAAGAGCGGGTGCTACTGGCGCTTCGACTGGTTCCTCAACGCGGACAACCCTCAAGTGAACTTCCGGCAGGTCTCGTGCCCTGCGGCCCTCACGTCTCGCAGCGGGTGTGTGAGGCAGGACAATGCCATGTTCAAATGA

[0051] Example 1

[0052] This example provides the effector VdGH45-1 to induce the ROS response in tobacco leaves, specifically as follows:

[0053] Using a 1 mL needleless syringe, approximately 20 µL of 5 µM VdGH45-1 protein was injected into the abaxial surface of 3- to 4-week-old tobacco leaves, with 1×PBS (pH 7.5) used as a negative control. Six hours after injection, the treated tobacco leaves were gently rinsed with deionized water, then immersed in DAB staining solution and incubated at 25°C in the dark for 8 hours. After incubation, the staining solution was removed, the leaves were rinsed with deionized water, and destained with 95% ethanol. Finally, the staining results were observed and photographed using a stereomicroscope.

[0054] The results are as follows Figure 1 As shown, significant reactive oxygen species (ROS) accumulation was observed in the injection area of ​​tobacco leaves injected with VdGH45-1 protein, while very weak ROS accumulation was observed in the injection area of ​​control leaves injected with 1×PBS.

[0055] The above experimental results show that the VdGH45-1 protein can effectively induce the outbreak of reactive oxygen species (ROS) in tobacco leaves.

[0056] Example 2

[0057] This embodiment demonstrates how the effector VdGH45-1 induces a resistance response in tobacco to Botrytis cinerea, and the specific process is as follows:

[0058] 20 µL of 5 µM VdGH45-1 effector protein was drawn into 4-week-old tobacco leaves via the abaxial surface using a sterile syringe. 20 µL of 5 µM GFP was used as a negative control. Eighteen h after injection, the injection site was punctured with a needle, and Botrytis cinerea cakes were collected and placed directly above the puncture site. The cakes were then incubated at 25°C and 80% humidity for 5 days. The diameter of the lesions in each treatment was observed and measured, and biomass was assessed. Results are as follows: Figure 2 and Figure 3 As shown: The area of ​​lesions on the experimental leaf was approximately 6.36 mm. 2 The area of ​​lesions on the control leaf was approximately 35.2 mm. 2 The lesion area in the experimental group was only 18.07% of that in the control group; the biomass of Botrytis cinerea also showed that the experimental group was only about 1 / 5 of that in the control group.

[0059] The above results indicate that VdGH45-1 protein can induce a resistance response in tobacco to Botrytis cinerea, and both the lesion area and pathogen biomass are significantly reduced.

[0060] Example 3

[0061] This example demonstrates that transient transformation of VdGH45-1 induces the expression of resistance-related genes in tobacco leaves. The specific process is as follows:

[0062] Four-week-old tobacco plants were selected, and the concentration of Agrobacterium pGR107-VdGH45-1 transiently transformed with VdGH45-1 was adjusted to OD600 0.5-0.8. 200 µL of Agrobacterium solution was injected from the underside of the leaves using a 1 mL syringe (needle removed). Agrobacterium of the same concentration transiently expressing pGR107-GFP was used 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 level of resistance-related genes.

[0063] The results are as follows Figure 4 As shown (*** indicates the pGR107-VdGH45-1 injection group, and the unmarked group is the pGR107-GFP injection control group), compared with the control group, the transcription level of disease resistance-related genes in the region of tobacco leaves injected with pGR107-VdGH45-1 was significantly increased.

[0064] The above experiments show that VdGH45-1 can induce the expression of resistance genes in tobacco leaves.

[0065] As demonstrated in Examples 1-3, VdGH45-1 elicits an immune response in a relatively short time; a strong reactive oxygen species burst is triggered as early as 6 hours after protein injection. Furthermore, this effector elicits a strong and rapid immune response even at a low concentration (5 µM). Therefore, compared to other identified effectors, VdGH45-1 can induce plant defense responses more quickly and effectively, thereby enhancing plant resistance.

[0066] 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.

[0067] 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.

[0068] 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 VdGH45-1 protein in enhancing plant resistance and / or inducing plant defense responses, characterized in that, The amino acid sequence of the VdGH45-1 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 Staphylococcus aureus and Verticillium dahliae; The induced plant defense response is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, namely Staphylococcus aureus and Verticillium dahliae. The plant in question is tobacco.

2. The application according to claim 1, wherein, The concentration of the VdGH45-1 protein used is 2-10 μM.

3. The application according to claim 2, wherein, The concentration of the VdGH45-1 protein used is 4-6 μM.

4. A method for enhancing plant resistance and / or inducing plant defense responses, characterized in that, The method includes the following steps: Apply the VdGH45-1 protein to the target plant, and / or introduce the gene encoding the VdGH45-1 protein into the target plant; The amino acid sequence of the VdGH45-1 protein is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the VdGH45-1 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 Staphylococcus aureus and Verticillium dahliae; The induced plant defense response is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, namely Staphylococcus aureus and Verticillium dahliae. The plant in question is tobacco.

5. The method according to claim 4, wherein, The gene encoding the VdGH45-1 protein was introduced into the target plant via a plant expression vector.

6. The method according to claim 5, wherein, The plant expression vector was pGR107.

7. The application of VdGH45-1 protein in pesticide preparation, characterized in that, The pesticide is used to enhance plant resistance and / or induce a defensive response in plants; The amino acid sequence of the VdGH45-1 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 Staphylococcus aureus and Verticillium dahliae; The induced plant defense response is: to induce a reduction in the damage to the plant caused by at least one of the pathogens, namely Staphylococcus aureus and Verticillium dahliae. The plant in question is tobacco.

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