Novel non-enzyme activity dependent plant immune activator protein NfXYG1 mutant E133A and application thereof

Abstract

The invention relates to the field of molecular biology, and particularly discloses a novel non-enzyme activity dependent type plant immune activator protein NfXYG1 mutant E133A and an application of the novel non-enzyme activity dependent type plant immune activator protein NfXYG1 mutant E133A. According to the NfXYG1 mutant E133A, the 133 catalytic key glutamic acid residue is replaced with alanine by adopting a site-specific mutagenesis method, and the amino acid sequence of the NfXYG1 mutant E133A is shown as SEQ ID NO.1. The NfXYG1 mutant E133A can still efficiently activate a plant immune pathway and induce broad-spectrum disease resistance while losing xyloglucan hydrolytic activity. The mutant provided by the invention retains a high protein expression quantity similar to that of a wild type, and has a unique non-enzymatic activity immune activation mechanism. Tests prove that the mutant can induce a tobacco necrosis reaction and has a remarkable effect on prevention and treatment of rice blast.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to a novel non-enzyme activity-dependent plant immune activation protein NfXYG1 mutant E133A and application thereof. BACKGROUND

[0002] Plant diseases are a key factor threatening global agricultural production, causing huge economic losses every year. Traditional chemical pesticides are prone to cause environmental pollution, pathogen resistance and agricultural product residues while preventing and treating diseases. The biological control technology based on plant immune activation proteins has become a new research direction due to its environmental friendliness and persistence. Among them, the glycoside hydrolase effectors from microorganisms are considered as the core components of new plant immune inducers because they can directly trigger the PTI / ETI cascade reaction in plants.

[0003] In the glycoside hydrolase GH12 family, some xyloglucanases (such as XEG1, BcXYG1 and VdEG1) exhibit a unique non-enzyme activity-dependent immune activation function. They can act as recognition effectors of pathogen-associated molecular patterns (PAMPs) and effectively activate plant immune responses. This feature not only enables them to maintain functional stability in complex environments, but also enhances plant disease resistance in cooperation with other substances. However, not all GH12 family proteins have a clear non-enzyme activity-dependent immune activation mechanism. More importantly, these effector proteins generally have a technical bottleneck of low expression level in heterologous expression systems, which seriously restricts their industrial application. Therefore, developing new proteins with high non-enzyme activity-dependent immune activation function and high-level recombinant expression characteristics has become an urgent need for current research. SUMMARY

[0004] In the application, the original sequence of the wild-type NfXYG1 gene is derived from the thermophilic fungus P1 strain isolated from acid mine drainage in Yunnan Province, which is identified as Neosartorya fischeri P1 strain by ITS sequence analysis. Neosartorya fischeri, and was deposited at China General Microbiological Culture Collection Center (CGMCC, Beijing, China) on September 3, 2013, and was assigned accession number CGMCC No. 3.15369. The strain is available to the public from the CGMCC since the date of deposit. The CGMCC website is https: / / cgmcc.net / , and the public can directly order the strain on the website. The E133A mutant is obtained by mutating glutamic acid (Glu) at position 133 of wild-type NfXYG1 to alanine (Ala) through site-directed mutagenesis technology. The function of the E133A mutant is verified through Escherichia coli and tobacco heterologous expression systems, and the E133A mutant does not depend on the characteristics of the original strain. The amino acid sequence of the wild-type NfXYG1 can be obtained from a known sequence in a public database (NCBI Reference Sequence: XP_001265191.1).

[0005] It is generally believed that destroying the catalytic key site may lead to complete inactivation of the protein, but the E133A mutant can efficiently activate the plant immune response and induce broad-spectrum disease resistance while completely losing the hydrolysis activity of xyloglucan. This result indicates that the wild-type NfXYG1 itself may have a unique non-enzyme activity-dependent immune activation mechanism, and the immune triggering function of the wild-type NfXYG1 does not depend on the catalytic activity, but is mediated by an unbroken domain or a specific recognition pattern. This finding provides a theoretical basis for developing a new generation of plant immune elicitors that do not depend on enzyme activity.

[0006] The specific technical solutions of the present application are as follows: The first aspect provides a novel non-enzyme activity-dependent plant immune activation protein NfXYG1 mutant protein E133A, which is obtained by mutating the key catalytic residue glutamic acid (Glu) at position 133 of the wild-type NfXYG1 to alanine (Ala) through rational design of proteins, and the amino acid sequence is SEQ ID NO. 1.

[0007] The second aspect provides an application of the mutant protein, and the application does not depend on the catalytic function of the mutant protein, and is any one of the following: A1) triggering a necrotic response in tobacco; A2) preparing a product for improving the immunity of tobacco to infestation by pests and diseases; A3) improving the resistance of rice to rice blast; A4) preparing a product for improving the resistance of rice to rice blast; In order to facilitate the purification or detection of the mutant protein, a tag protein can be connected to the amino terminal or carboxyl terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing.

[0008] The tag protein includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0009] The third aspect provides an application of the biological material related to the mutant protein, which does not depend on the catalytic function of the mutant protein, and is any one of the following: D1) inducing a necrotic response in tobacco; D2) preparing a product for improving the immunity of tobacco to insect and disease infection; D3) improving the resistance of rice to rice blast; D4) preparing a product for improving the resistance of rice to rice blast; The biological material is any one of the following E1) to E4): E1) a nucleic acid molecule encoding the mutant protein described above; E2) an expression cassette containing the nucleic acid molecule of E1); E3) a recombinant vector containing the nucleic acid molecule of E1), or a recombinant vector containing the expression cassette of E2); E4) a recombinant microorganism containing the nucleic acid molecule of E1), or a recombinant microorganism containing the expression cassette of E2), or a recombinant microorganism containing the recombinant vector of E3).

[0010] Further, the nucleotide sequence encoding the mutant protein is shown in SEQ ID NO. 2.

[0011] The fourth aspect provides a method for inducing a necrotic response in tobacco, comprising: treating tobacco leaves with the biological material to achieve a necrotic response in tobacco leaves.

[0012] The fifth aspect provides a method for improving the resistance of rice to rice blast, comprising: treating rice leaves with the mutant protein to achieve an improvement in the resistance of rice to rice blast.

[0013] Advantages of the present application: The xyloglucanase NfXYG1 mutant E133A of the present application is obtained by rational design of protein, in which the catalytic residue glutamic acid (Glu) at position 133 of wild-type NfXYG1 is mutated to alanine (Ala). Although it is conventionally expected that this mutation will result in complete loss of protein function, E133A still retains immune activation ability under the condition of enzyme activity loss, which reveals that wild-type NfXYG1 may have a unique non-enzyme activity-dependent immune activation mechanism. The mutant protein not only retains the advantages of high expression amount of wild-type protein and environmental friendliness, but also significantly improves the immune induction efficiency through a unique non-enzyme activity-dependent mechanism. Experiments have confirmed that the mutant has a significant effect on the prevention and treatment of rice blast in rice, and exhibits important application value in green prevention and control of crops. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 NfXYG1 and mutant E133A induced necrosis phenotype in tobacco: BcXYG1 19-248 NfXYG1 19-238 and enzyme activity mutant NfXYG1 19-238 -E133A transiently expressed in tobacco, all caused cell necrosis.

[0015] Figure 2 NfXYG1 and mutant E133A protein penetration treatment of tobacco phenotype analysis: A, chloronitro tetrazolium blue staining (NBT staining); B, diaminobenzidine staining (DAB staining).

[0016] Figure 3 NfXYG1-E133A protein rice blast prevention and treatment effect diagram. DETAILED DESCRIPTION

[0017] The specific embodiments of the present application are described below to facilitate understanding of the present application by those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments. For those skilled in the art, any changes that do not exceed the spirit and scope of the invention defined by the claims are within the scope of the present patent protection; all embodiments based on the concept of the present application should be protected.

[0018] Explanation of the sequence listing of the present application: The amino acid sequence of NfXYG1-E133A is shown in SEQ ID NO. 1: MKFSLSVALSLAAATAQAATQFCDQWGSVTEGNYILYNNLWGQAQATSGSQCTTFESLS GNTIVWNTKWSWSGGQGQVKSFANAALQFTPKKLSSVKSIDSTWTWNYSGSNIVAD VAYDMFLSTSPGGDHNYAIMVWLGALGGAGPISSTGSPIATPTVAGIKFNLYLGPNGSM QVYSFVAQSTTKSFSGDMRDFFTYLEGNQGLSSDLYLVDVQAGTEPFSGSNAVFTVSD YSVSA The nucleotide sequence of NfXYG1-E133A is shown as SEQ ID NO. 2: ATGAAGTTCAGCCTCTCTGTCGCCCTCTCGCTCGCCGCAGCGACTGCCCAGGCAGCCACCCAGTTCTGTGACCAATGGGGCTCGGTCACCGAGGGCAACTACATTCTTTACAACAATCTTTGGGGTCAGGCCCAGGCCACCTCCGGCTCCCAATGCACCACCTTTGAATCTCTTTCGGGCAACACGATCGTCTGGAACACCAAGTGGTCTTGGTCCGGTGGCCAGGGCCAGGTCAAGAGCTTCGCCAATGCCGCTCTGCAGTTCACCCCCAAGAAACTGAGCAGCGTCAAGAGCATTGACTCGACTTGGACGTGGAACTACTCTGGCTCCAACATCGTCGCCGATGTCGCCTACGACATGTTCCTCAGCACCTCCCCCGGCGGCGACCACAACTACGCGATCATGGTCTGGCTAGGTGCTTTGGGCGGCGCCGGTCCCATTTCCTCGACGGGCTCGCCCATCGCCACGCCTACCGTCGCTGGCATCAAGTTTAACCTGTACCTCGGCCCGAACGGCTCGATGCAGGTGTACAGCTTCGTGGCCCAGTCGACCACGAAGAGCTTCTCCGGCGACATGCGCGACTTCTTCACGTACCTGGAGGGCAACCAGGGACTGTCCAGCGACCTGTACCTGGTCGACGTGCAAGCCGGTACCGAGCCCTTCAGCGGAAGCAATGCCGTTTTCACGGTATCCGACTACTCTGTCAGTGTTGCCTAA The signal peptide sequence of tobacco PR1a is shown in SEQ ID NO. 3: ATGGGATTTGTTCTCTTTTCACAATTGCCTTCATTTCTTCTTGTCTCTACACTTCTCTTATTCCTAGTAATATCCCACTCTTGCCGTGCCCAAAAT Example 1 Prokaryotic expression system (1) Construction of prokaryotic expression vector The wild type NfXYG1 gene is 717 bp in length (including signal peptide), encodes 238 amino acids, and the mature protein has a molecular weight of 25.2 kDa. It has a typical β-jelly-roll structure of GH12 family, and the enzyme activity catalytic sites are E133 and E219.

[0019] The 19-238 amino acids of the original sequence of NfXYG1 were codon-optimized E coli and inserted into the pET-22b vector Amp , and the enzyme cleavage site Eco RI— Not I, and the N-terminal carries a 6×His tag. The gene was synthesized by Anhui General Biotech Co., Ltd. On this basis, the enzyme activity catalytic site E133 was mutated using a point mutation kit, transformed into E. coli BL21 (DE3) competent cells, and positive clones were selected for bacterial liquid PCR and agarose gel electrophoresis verification. The correct strain was sent to Huada Biological Technology Co., Ltd. for sequencing.

[0020] (2) Induction expression of recombinant protein The sequenced BL21 (DE3) strain was inoculated into 30 mL of LB liquid medium containing Kan ＋ (50 mg / mL) at a concentration of 1%, and incubated at 37°C, 220 rpm, and overnight.

[0021] The activated strain was inoculated into 300 mL of LB liquid medium (1L conical flask) at a concentration of 1%, and Kan ＋ (50 mg / mL) was added at a ratio of 1‰. The culture was incubated at 37°C, 220 rpm, for 4 h, and the OD600 value was measured. OD 600 The value was between 0.4 and 0.8 (about 0.6).

[0022] 60 μL of isopropyl-β-D-thiogalactoside (1 mol / L) was added to every 100 mL of LB at a ratio of 60 μL of isopropyl-β-D-thiogalactoside, and the culture was induced at 37°C, 220 rpm, for 4-6 h.

[0023] After induction, the culture was centrifuged at 4°C, 12000 rpm, for 15 min, and the supernatant was discarded.

[0024] The bacterial pellet was resuspended in 10 mL of sterile water and transferred to a 50 mL centrifuge tube, which was stored at 4°C.

[0025] The resuspended bacterial pellet was placed in an ice water bath and sonicated using an ultrasonic cell disruptor. The sonication program was as follows: amplitude 6, power ratio 40%, sonication on 6.0 s, sonication off 10 s, and total time 25 min.

[0026] After the completion of the break, 4℃, 12000 rpm, centrifugation for 20 min, transfer the supernatant to a new 50 mL centrifuge tube is the crude protein, stored at 4℃.

[0027] Example 2 Plant transient expression vector construction The tobacco PR1a signal peptide sequence used in the application is shown in SEQ ID NO. 3.

[0028] The vector used is pBI121-eGFP, Kan ＋ resistance, containing eGFP tag at the C terminal, using this skeleton to construct PR1a fusion protein gene.

[0029] The NfXYG1 self signal peptide (first 18 amino acids) is replaced with tobacco PR1a signal peptide sequence, and inserted into the vector pBI121-eGFP, enzyme cutting site Xba I— Xma I, the gene synthesis is synthesized by Anhui General Biological Company, to obtain pBI121-PR1a-NfXYG1 19-238 -eGFP vector.

[0030] Using pBI121-PR1a-NfXYG1 19-238 -eGFP as a template, using specific primers, the 133th amino acid of the original sequence is mutated to alanine (Ala), to obtain the enzyme activity deletion vector pBI121-PR1a-NfXYG1 19-238 -E133A-eGFP, the gene synthesis is synthesized by Anhui General Biological Company.

[0031] The typical xyloglucanase immunoprotein BcXYG1 is used as a positive control, the 19-248 amino acid sequence of the gene is cloned without signal peptide, and the PR1a signal peptide sequence is also fused, the vector backbone is pBI121-eGFP, enzyme cutting site Xba I— Xma I, the gene synthesis is synthesized by Anhui General Biological Company, to obtain the positive vector pBI121-PR1a-BcXYG1 19-248 -eGFP.

[0032] The tobacco PR1a signal peptide is combined to the pBI121-eGFP skeleton, enzyme cutting site Xba I— Xma I, to obtain the vector pBI121-PR1a-eGFP, as a negative control.

[0033] Example 3 Tobacco leaf necrosis reaction The obtained positive transformants were inoculated into LB liquid medium and cultured at 28°C with 180 rpm for 24 h. The bacterial solution was transferred into a 50 mL centrifuge tube and centrifuged at 6000 rpm for 10 min. The supernatant was discarded and the bacterial cells were collected. 5 mL of Agrobacterium suspension containing 1% 2-ethanesulfonic acid, 1% magnesium chloride, and 0.2% acetyl-syringone was taken and used to further blow the bacterial cells to complete dispersion without clumping. The concentration of the bacterial solution was adjusted to OD 600 = 0.5-0.6.

[0034] After the needle of a disposable sterile syringe was removed, 20 μL of the bacterial solution was injected into the middle leaf of tobacco at the 5-6 leaf stage by pressure. The phenotype of the injected leaf was observed every day after 36 h.

[0035] As Figure 1 , the NfXYG 19-238 and the NfXYG 19-238 Necrotic spots were observed at the infiltration points of the E133A mutant, and the phenotype was consistent with the positive control, while no necrotic spots were generated in the negative control. This result indicates that even if the self signal peptide is deleted, NfXYG1 can still be secreted to the apoplast by the tobacco signal peptide, thereby triggering hypersensitive necrosis of tobacco cells. In addition, the enzyme activity mutant E133A also retains the ability to induce hypersensitive necrosis of cells, but it needs to be secreted to the apoplast space by the signal peptide.

[0036] Example 4 ROS detection of tobacco leaves To further verify the ROS burst generated when the E133A mutant triggers plant necrosis, superoxide anion and hydrogen peroxide were stained using nitro blue tetrazolium chloride staining solution and diaminobenzidine staining solution, respectively. The specific steps are as follows: The tobacco leaves treated with the purified protein infiltration were cut and completely immersed in a 8 cm diameter culture dish containing 0.1% nitro blue tetrazolium chloride staining solution (or 1.0 mg / mL diaminobenzidine staining solution) at 25°C in the dark for 40 min. The leaves were taken out and placed in 95% ethanol, and the decolorization treatment was repeated three times for 10 min in a boiling water bath until the chlorophyll was completely removed. The decolorized leaves were fixed and stored with 95% ethanol for subsequent photography and observation.

[0037] As Figure 2 , the NfXYG 119-238 and the NfXYG 19-238Cell necrosis induced by the E133A mutant appears as yellow necrotic spots that cannot be identified by nitro blue tetrazolium chloride staining solution and diaminobenzidine staining solution. A burst of reactive oxygen species occurs at the junction of the necrotic area and healthy tissue, showing a large amount of brown precipitate. This result shows that the E133A mutant and wild-type NfXYG1 induce plant cell death in a similar way, which means that the ability of NfXYG1 to induce plant cell death is independent of its xyloglucanase activity. This means that NfXYG1 may have a unique protein domain or amino acid sequence that is involved in regulating cell death.

[0038] Example 5 Effect of Controlling Rice Blast Use Nipponbare ( Nipponbare , NPB), isolated races of rice blast fungus ( Magnaporthe oryzae isolateRB22) was used for a preventive drug experiment with four treatment groups: (1) Spray 5 mL of 500-fold diluted E133A mutant protein solution on the leaves of rice at the four-leaf and one-heart stage. 24 hours later, spray 10 mL of rice blast fungus spore suspension (2.5×10 5 spores / mL).

[0039] (2) Spray 5 mL of 1000-fold diluted E133A mutant protein solution on the leaves of rice at the four-leaf and one-heart stage. 24 h later, spray 10 mL of rice blast fungus spore suspension (2.5×10 5 spores / mL).

[0040] (3) Spray 5 mL of water on the rice leaves, and 24 hours later spray 10 mL of rice blast fungus spore suspension (2.5×10 5 spores / mL).

[0041] (4) Spray 5 mL of water on the rice leaves.

[0042] A completely randomized block design was used for each group, with three biological replicates. Rice seedlings in each treatment group were placed in a large storage box, separated by black plastic bags. Each treatment group had four small rice pots, with nine rice plants per pot (3 × 3). Seven days after spraying with the rice blast solution, diseased leaves were cut and photographed for lesions.

[0043] like Figure 3 Both high and low concentrations of the E133A mutant protein solution induced the expression of rice defense mechanism genes, enhancing resistance to pathogen infection. Furthermore, as the concentration increased, its effectiveness in preventing rice blast infection improved.

Claims

1. An application of a mutant protein E133A of xyloglucanase NfXYG1, characterized in that: The application is independent of the catalytic function of the mutant protein E133A and is any of the following: A1) stimulate tobacco necrosis reaction; A2) Preparation of products to enhance tobacco's immunity to pests and diseases; A3) Improve rice blast resistance; A4) preparing products for improving rice blast resistance; The mutant protein E133A is a protein with an amino acid sequence of SEQ ID NO.

1.

2. Use of a biomaterial related to the mutant protein E133A according to claim 1, characterized in that: The application is independent of the catalytic function of the mutant protein E133A and is any of the following: D1) stimulate tobacco necrosis reaction; D2) Preparation of products to enhance tobacco's immunity to pests and diseases; D3) Improve rice blast resistance; D4) Preparation of products for improving rice blast resistance; The biological material is any one of the following E1) to E4): E1) a nucleic acid molecule encoding the protein according to claim 1; E2) an expression cassette containing the nucleic acid molecule described in E1); E3) a recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2); E4) A recombinant microorganism containing the nucleic acid molecule of E1), or a recombinant microorganism containing the expression cassette of E2), or a recombinant microorganism containing the recombinant vector of E3).

3. The use according to claim 2, characterized in that The nucleotide sequence encoding the mutant protein E133A is shown in SEQ ID NO.

2.

4. A mutant protein E133A of xyloglucanase NfXYG1, characterized in that The mutant protein E133A is a protein with an amino acid sequence of SEQ ID NO.

1.

5. The mutant protein E133A according to claim 4, characterized in that The mutant protein E133A is an enzyme-independent protein.

6. A method for stimulating tobacco necrosis reaction, comprising: The tobacco leaves are treated by infiltration using the biological material described in claim 2.

7. A method for improving rice blast resistance, characterized in that: The mutant protein E133A according to claim 5 is used to spray the surface of rice leaves.

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