Plant immunity elicitor protein PpAE4 and application thereof

Abstract

The application discloses a plant immunity elicitor protein PpAE4 and application thereof, the protein can induce plant resistance, and can be applied to the field of plant disease prevention and treatment as a plant immunity activator. The amino acid sequence of the protein is shown as SEQ ID NO:2. High-concentration protein can be obtained by using an engineering strain for expression, and the protein can be used for treating plants to improve the disease resistance of the plants to soybean rust fungi, and the required concentration is low, the effect is quick, and the action time is long. The PpAE4 provides a new way for improving the disease resistance of plants and preventing and treating plant diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a plant immune-inducing protein PpAE4 and its applications. Background Technology

[0002] *Phakopsora pachyrhizi*, a basidiomycete fungus, has a wide host range, infecting 149 species of leguminous plants in 49 genera. At least 30 species across 17 genera are known as natural hosts, with *Glycine* being the most important. Soybean rust (ASR) caused by *P. pachyrhizi* has resulted in significant economic losses and poses a potential threat to food security. Due to the scarcity of resistant resources in soybeans, current control of soybean rust relies primarily on fungicides. For example, in Brazil, the cost of controlling the disease reached $2 billion in 2003. The massive use of chemical fungicides and unscientific application methods have led to a series of problems, including pesticide ineffectiveness, environmental pollution, and crop damage. For instance, pesticides for controlling soybean rust include triazoles, methoxyacrylates, and pyrazolamides, but their efficacy has significantly decreased. For example, in Brazil, fungicides that previously achieved 90% control efficacy have now seen their efficacy drop to 20%–40%. Specifically, the efficacy of prothioconazole has decreased from 100% to around 80%; the efficacy of methoxyacrylate fungicides has generally declined, with azoxystrobin and pyraclostrobin even dropping to 30%–40%, showing almost no effect in field observations; the efficacy of pyrazoleamide fungicides such as benzo[a]flufenicol has also decreased.

[0003] Previous studies have shown that soybeans generate an immune response in the early stages of rust fungal infection. Therefore, developing effective plant immune-inducing proteins can provide effective protein resources for the development of novel protein-based biopesticides that activate plant immunity, which is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a plant immune-inducing protein PpAE4 and its applications.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] In a first aspect, the present invention seeks protection for the use of the plant immune-inducing protein PpAE4 in any of the following:

[0007] (a1) Improve plant disease resistance;

[0008] (a2) Prepare products to improve plant disease resistance;

[0009] (a3) Inhibits the growth and development of soybean rust fungus;

[0010] (a4) Prepare products for inhibiting the growth and development of soybean rust fungus;

[0011] (a5) Induces the release of reactive oxygen species in plants;

[0012] (a6) Prepare products for inducing the release of reactive oxygen species in plants;

[0013] (a7) Induces plant defense responses;

[0014] (a8) Prepare products for inducing plant defense responses;

[0015] The plant immune-inducing protein PpAE4 is as follows (b1) or (b2):

[0016] (b1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0017] (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0018] Secondly, the present invention seeks protection for the use of biological materials related to the aforementioned plant immune-inducing protein PpAE4 in any of the following:

[0019] (a1) Improve plant disease resistance;

[0020] (a2) Prepare products to improve plant disease resistance;

[0021] (a3) Inhibits the growth and development of soybean rust fungus;

[0022] (a4) Prepare products for inhibiting the growth and development of soybean rust fungus;

[0023] (a5) Induces the release of reactive oxygen species in plants;

[0024] (a6) Prepare products for inducing the release of reactive oxygen species in plants;

[0025] (a7) Induces plant defense responses;

[0026] (a8) Prepare products for inducing plant defense responses;

[0027] The biomaterial is at least one of the following (c1)-(c4):

[0028] (c1) The nucleic acid molecule encoding the aforementioned plant immune-inducing protein PpAE4;

[0029] (c2) An expression cassette containing the nucleic acid molecule described in (c1);

[0030] (c3) A recombinant vector containing the nucleic acid molecule described in (c1) or a recombinant vector containing the expression cassette described in (c2);

[0031] (c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3).

[0032] Furthermore, the nucleic acid molecule is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1.

[0033] Furthermore, the plants are soybeans, alfalfa, and tomatoes; the disease resistance in (a1) and (a2) refers to resistance to soybean rust fungus.

[0034] Furthermore, the above-mentioned application involves spraying the plant immune induced protein PpAE4 onto the plant surface or injecting it into plant leaves; or spraying the recombinant protein of the plant immune induced protein PpAE4 obtained by expressing and purifying (c1) the nucleic acid molecule onto the plant surface or injecting it into plant leaves. Preferably, the concentration of the sprayed plant immune induced protein PpAE4 is 0.8–1.5 μM.

[0035] Thirdly, the present invention claims protection for a method for improving plant disease resistance, which involves spraying the plant immune-inducing protein PpAE4 onto the plant surface or injecting it into the plant leaves; or spraying or injecting a recombinant protein of the plant immune-inducing protein PpAE4 obtained by expressing and purifying the nucleotide sequence of a nucleic acid molecule as shown in SEQ ID NO:1 onto the plant surface or into the plant leaves; wherein the plant immune-inducing protein PpAE4 is as follows (b1) or (b2):

[0036] (b1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0037] (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1);

[0038] The disease resistance mentioned refers to resistance to soybean rust fungus; the plants mentioned are soybean, alfalfa, and tomato.

[0039] Preferably, the concentration of the sprayed plant immune-inducing protein PpAE4 is 0.8–1.5 μM.

[0040] Fourthly, the present invention claims protection for a plant immune-inducing protein PpAE4, which is as follows (b1) or (b2):

[0041] (b1) A protein with the amino acid sequence shown in SEQ ID NO:2;

[0042] (b2) A fusion protein with the same function obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein described in (b1).

[0043] Fifthly, the present invention seeks protection for biological materials related to the plant immune-inducing protein PpAE4, wherein the biological material is at least one of the following (c1)-(c4):

[0044] (c1) The nucleic acid molecule encoding the plant immune-inducing protein PpAE4;

[0045] (c2) An expression cassette containing the nucleic acid molecule described in (c1);

[0046] (c3) A recombinant vector containing the nucleic acid molecule described in (c1) or a recombinant vector containing the expression cassette described in (c2);

[0047] (c4) A recombinant microorganism containing the nucleic acid molecule described in (c1), or a recombinant microorganism containing the expression cassette described in (c2), or a recombinant microorganism containing the recombinant vector described in (c3).

[0048] In a specific embodiment of the present invention, the recombinant vector is a eukaryotic expression vector obtained by inserting the PpAE4 encoding gene into pPIC9K. This recombinant vector is introduced into the yeast strain *P. pastoris* KM71 for expression, yielding a fusion protein (HIS-PpAE4) with a molecular weight of approximately 15 kDa. Experiments show that this recombinant protein can induce resistance responses in plants such as soybean, alfalfa, and tomato, thereby enhancing plant resistance to soybean rust fungus and alleviating related disease symptoms.

[0049] Studies have shown that the plant immune-inducing protein PpAE4 described in this invention can effectively induce the crucial defense response of reactive oxygen species (ROS) bursts. Theoretically, for plants capable of recognizing this protein, PpAE4 can induce a defense response or enhance disease resistance. In specific implementation cases, its effectiveness on soybeans, alfalfa, and tomatoes has been successfully verified, particularly significantly enhancing resistance to soybean rust fungus.

[0050] The beneficial effects of this invention are as follows:

[0051] The plant immune-inducing protein of this invention can significantly improve plant disease resistance, requires low concentration, is fast-acting, and has a long duration of action. PpAE4 activates the plant's own immune system, providing a new approach to improving plant resistance, and therefore has broad application prospects in agricultural production. Attached Figure Description

[0052] Figure 1PpAE4 protein was expressed in yeast strain P. pastoris KM71, and the antibody used for Western blot detection was anti-HIS.

[0053] Figure 2 To detect reactive oxygen species (ROS) bursts induced by the plant immune-inducing protein PpAE4 in soybeans, alfalfa, and tomatoes.

[0054] Figure 3 This study aimed to induce resistance in soybean to soybean rust fungus using the plant immune-inducing protein PpAE4. A represents the infection symptoms of soybean rust fungus in soybeans treated with and untreated with PpAE4 protein; B represents the spore mass statistics of soybeans treated with and untreated with PpAE4 protein after inoculation with soybean rust fungus; and C represents the biomass detection of soybeans treated with and untreated with PpAE4 protein after inoculation with soybean rust fungus. Detailed Implementation

[0055] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. The primers involved in the embodiments of the present invention were synthesized by Sangon Biotech Co., Ltd.

[0056] The soybean rust fungus strain is Phakopsora pachyrhizi SS4, which is a standard strain widely known to those skilled in the art and used in research on soybean rust resistance.

[0057] The room temperature described in this invention is 25±10℃.

[0058] Example 1. Isolation and identification of plant immune-inducing protein PpAE4

[0059] A suspension of zoospores of *Solanum sarcodactylis* was prepared, and the inoculum concentration of *Solanum sarcodactylis* urediniospores was adjusted to 100 spores / µL. Soybean leaves were treated with Tween-20 at a concentration of 0.1% (v / v), followed by treatment with sterile distilled water to thoroughly remove the Tween-20. Soybean leaves were immersed in the urediniospore suspension for 30 min, then gently removed and placed in petri dishes lined with moistened filter paper to maintain humidity. The leaves were incubated at 25°C in the dark for 3 days. The intercellular fluid of the infected soybean leaves was collected using a vacuum pump and further separated and concentrated using ultrafiltration. The collected fluid was then sent to BGI Genomics in Shenzhen for proteomics analysis. Based on the proteomics data and comparison with the *Solanum sarcodactylis* protein database, the secreted protein PpAE4 of *Solanum sarcodactylis* was identified, and its amino acid sequence is shown in SEQ ID NO:2.

[0060] Example 2. Cloning of the gene encoding the plant immune-inducing protein PpAE4

[0061] (1) Total RNA extraction:

[0062] Mixed samples taken 1 day, 3 days, 5 days, 7 days and 14 days after infection of the susceptible soybean rust cultivar Williams 82 by urediniospores were used as materials. Total RNA was extracted using the Omega RNA Extraction Kit according to the instructions, and the RNA content and quality were detected by spectrophotometer.

[0063] (2) Reverse transcription to generate the first strand:

[0064] Use 0.7 μg of RNA as a template and synthesize cDNA according to the instructions for use of Takara PrimeScript reverse transcriptase reagent. Adjust the volume to 20 μL. Use an appropriate amount of the reverse transcription product for subsequent gene cloning PCR.

[0065] (3) Using the first strand of cDNA as a template, primers were designed to amplify the full length of the PpAE4 encoding gene by PCR:

[0066] PCR amplification primer sequences:

[0067] Upstream primer: 5'-CAGCTAGCATCGATTCCCGGGATGTCTTTTACAAAAAAGG-3'

[0068] Downstream primer: 5'-AATCTCTAGAGGATCCCCGATCTTAATGACATTCACAG-3',

[0069] The 50 μL reaction system consisted of: 10 μL of 5×buffer, 4 μL of 2.5 mM dNTPs, 0.5 μL of Takara PrimerSTARTaq enzyme, 1 μL of template cDNA, and water to a final volume of 50 μL.

[0070] The PCR amplification program was as follows: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, repeated 35 times, with a final extension at 72℃ for 10 min. Electrophoresis was performed on an agarose gel, followed by ethidium bromide (EB) staining and photographing. The results were recorded, and the PCR product encoding the PpAE4 gene was recovered by gel excision. The electrophoretic bands were recovered using the Agarose Gel DNA Purification Kit (TaKaRa). The PCR product of the PpAE4 encoding gene recovered from gel excision was ligated into the SmaI-digested pGR107::3HA vector (purchased from BioVector Plasmid Vector Culture Collection Center) according to the instructions of the CloneExpressII One Step Cloning Kit (Vazyme) to obtain the pGR107::PpAE4-3HA plasmid. The plasmid was transformed into E. coli competent cells JM109 and cultured in the dark at 37 °C for 16 h on LB agar plates containing 50 µg / mL kanamycin. Three positive clones were selected for colony PCR verification, and the plasmid was extracted according to the plasmid extraction kit (Takara). The plasmid was sent to Sangon Biotech Co., Ltd. for sequencing. The sequence obtained should be consistent with the sequence of SEQ ID NO:1.

[0071] Example 3. Eukaryotic expression and purification of plant immune-inducing protein PpAE4

[0072] (1) Construction of eukaryotic expression vectors

[0073] Specific primers were designed to amplify the PpAE4 gene (SEQ ID NO:1), and the gene was constructed into the eukaryotic expression vector pPIC9K via the Snabl restriction site. The specific primer sequences are as follows:

[0074] Upstream primer: 5'-AGAGAGGCTGAAGCTTACGCCTTGGTTCAACCTAGGG-3'

[0075] Downstream primer: 5'-CGCCCTAGGGAATTCTACTCAGTGGTGGTGGTGGTGGTGGATCTT

[0076] AATGACATTCACAG-3'

[0077] (2) Expression and purification of PpAE4 protein

[0078] The constructed pPIC9K recombinant vector was transformed into yeast strain P. pastoris KM71; single colonies were picked, and colony PCR was performed using the primers for the following PpIC9K vector to screen for positive clones.

[0079] pPIC9K-seq-F:GACTGGTTCCAATTGACAAGC

[0080] pPIC9K-seq-R:CCTCTTGATTAGAATCTAGCA

[0081] The specific expression and purification steps are as follows:

[0082] 1) Pick a single positive clone and add it to 2 mL of YPD liquid medium for initial culture. Incubate overnight at 200 rpm in a shaker at 30°C.

[0083] 2) The next morning, add the bacterial culture to Buffered Glycerol-complex Medium (BMGY) at a volume ratio of 1:100 for expansion culture, and incubate overnight at 200 rpm in a shaker at 30°C. On the afternoon of the third day, around 16:00-17:00, remove the culture medium from the shaker and let it stand for 3-4 hours.

[0084] 4) After standing, quickly discard the supernatant and add an equal volume of methanol-complex medium (BMMY) to induce protein expression.

[0085] 5) Add 1% filtered and sterilized methanol again the next day, and protein expression will continue for about 3 days.

[0086] 6) Collect the supernatant for Western blot analysis. If protein expression is observed, add the bacterial culture to a centrifuge bottle and centrifuge at 8000 rpm for 20 min. Collect the supernatant and add (NH4)2SO4 (500 g per 1 L of protein) to precipitate for 2-3 days.

[0087] 7) Discard the supernatant, leave a small amount of liquid to suspend the precipitate, centrifuge at 8000 rpm for 20 min, and discard the supernatant.

[0088] 8) Add 20 mL of ultrapure water to dissolve the protein, adjust the pH to approximately 7.4, and centrifuge at 4°C and 8000 rpm for 10 min. Collect the supernatant. Filter the supernatant into a 50 mL BD tube and perform nickel affinity chromatography using an AKTA purification system. Desalt the purified collection using a desalting column, then determine the protein concentration. Perform SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and Western blot analysis to detect the purified target protein.

[0089] Results: SDS-PAGE analysis revealed a recombinant PpAE4 protein (HIS-PpAE4) with an N-terminus HIS tag and a molecular weight of approximately 15 kDa. Figure 1 As shown.

[0090] Example 4. Plant immune-inducing protein PpAE4 induces plant immune responses in soybean, alfalfa and tomato.

[0091] Soybean, alfalfa, and tomato leaves were collected, and leaf discs with a diameter of 0.5 cm were obtained using a perforator. The leaf discs were placed in 96-well plates, and 200 µL of sterile water was added to each well. The plates were incubated overnight at room temperature to deplete endogenous reactive oxygen species (ROS) and induce stomatal opening. A reaction buffer containing luminol (35.4 µg / mL), horseradish peroxidase (10 µg / mL), and the purified protein (1 µM) prepared in Example 3 was prepared using sterile ddH2O. Under light-protected conditions, the sterile water in the wells was aspirated, and 200 µL of reaction buffer was added to each well. The luminescence signal was immediately measured using a GLOMAX96 microplate spectrophotometer (Promega, Madison, WI, USA) to quantify the ROS burst level.

[0092] Results: 1 µM PpAE4 protein induced reactive oxygen species (ROS) bursts in soybean, alfalfa, and tomato (e.g., ...). Figure 2 (As shown).

[0093] Example 5. Plant immune-inducing protein PpAE4 enhances disease resistance in soybeans.

[0094] 1 µM PpAE4 protein was sprayed onto soybean leaves, with BSA and chitin at the same concentration serving as negative and positive controls, respectively. After 24 h of protein treatment, soybean rust fungus was inoculated onto the leaves. Infection was observed, photographed, and the number of spore masses was counted 13 days after inoculation. Total RNA was extracted from leaf samples, and the relative biomass of soybean rust fungus was determined using quantitative real-time PCR. The α-tubulin gene of soybean rust fungus and the soybean internal reference gene Ubiquitin-3 were selected for relative quantitative analysis. The primers used for the quantitative real-time PCR reaction are as follows:

[0095] Soybean rust fungus α-tubulin:

[0096] qPCR-α-tubulin-F: 5'-CCAAGGCTTCTTCGTGTTTCA-3'

[0097] qPCR-α-tubulin-R: 5'-CAAGAGAAGAGCGCCAAACC-3'

[0098] Soy Ubiquitin-3:

[0099] qPCR-Ubiquitin-3-F: 5'-GTGTAATGTTGGATGTGTTCCC-3'

[0100] qPCR-Ubiquitin-3-R: 5'-ACACAATTGAGTTCAACACAAACCG-3'

[0101] Results: Compared with the negative control BSA protein treatment, the symptoms of soybean rust on soybean leaves treated with PpAE4 and the positive control chitin were significantly reduced (e.g., Figure 3 In A), the number of spore masses is significantly reduced (e.g. Figure 3 In the case of B), the biomass of soybean rust fungus infection is significantly reduced (e.g., Figure 3 (C in the middle).

[0102] sequence list

[0103] SEQ ID NO:1: Full-length nucleotide sequence of the gene encoding PpAE4, *Phakopsorapachyrhizi*, the causal agent of soybean rust, 366 bp in length.

[0104] ATGTCTTTTACAAAAAAGGTACTATTATCAATTTGCTTTTTTTGCCATTTTAACCATTTCAAAGGCCGCCTTGGTTCAACCTAGGGCCCGATGCTTAGGCGATAGTGGAATCATCGCGGACGACTGTAGGAGCGCTCTGTCTCAATTTCGTCTTGATCAGGGTGGATATGTTCAGTACAATACT CAAGGAGAACAAAGAGATTTTGGGACTTGTAGGATCCTATTAAAAAAGAGCGTAGCAGTATCCAACTCTGGACAAATTGCGCCTAACGACTTGGTTCAATTCGTTTCAAATAGTTTGGCGGCATGCAATGGAGGGACGGCAACTTTTGAAGTTCAAGATTTAACTGTGAATGTCATTAAGATC

[0105] SEQ ID NO:2: Full-length amino acid sequence of PpAE4, soybean rust fungus (Phakopsorapachyrhizi), sequence length 122 aa

[0106] MSFTKKVLLSICFFAILTISKAALVQPRARCLGDSGIIADDCRSALSQFRLDQGGYVQYNTQGEQRDFGTCRILLKKSVAVSNSGQIAPNDLVQFVSNSLAACNGGTATFEVQDLTVNVIKI.

Claims

1. Use of a plant immunity elicitor protein PpAE4 in any of the following: (al) improving the resistance of soybean to Phakopsora pachyrhizi; (a2) preparing a product for improving the resistance of soybean to Phakopsora pachyrhizi; (a3) inducing the active oxygen burst of soybean, alfalfa and tomato; (a4) preparing a product for inducing the active oxygen burst of soybean, alfalfa and tomato; wherein the plant immunity elicitor protein PpAE4 is (bl) or (b2): (bl) a protein having an amino acid sequence as shown in SEQ ID NO: 2; (b2) a fusion protein having the same function by linking a protein tag to the N-terminus and / or C-terminus of the protein of (bl); and wherein the use is spraying the plant immunity elicitor protein PpAE4 onto the surface of a plant. The concentration of the sprayed plant immunity elicitor protein PpAE4 is 0.8-1.5 μM.

3. Use of a biological material related to the plant immunity elicitor protein PpAE4 of claim 1 in any of the following: (al) improving the resistance of soybean to Phakopsora pachyrhizi; (a2) preparing a product for improving the resistance of soybean to Phakopsora pachyrhizi; (a3) inducing the active oxygen burst of soybean, alfalfa and tomato; (a4) preparing a product for inducing the active oxygen burst of soybean, alfalfa and tomato; wherein the biological material is at least one of (cl)-(c4): (cl) a nucleic acid molecule encoding the plant immunity elicitor protein PpAE4 having a nucleotide sequence as shown in SEQ ID NO: 1; (c2) an expression cassette containing the nucleic acid molecule of (cl); (c3) a recombinant vector containing the nucleic acid molecule of (cl), or a recombinant vector containing the expression cassette of (c2); (c4) a recombinant microorganism containing the nucleic acid molecule of (cl), or a recombinant microorganism containing the expression cassette of (c2), or a recombinant microorganism containing the recombinant vector of (c3); and wherein the use is spraying the recombinant protein of the plant immunity elicitor protein PpAE4 obtained by the biological material of (cl)-(c4) onto the surface of a plant. The concentration of the sprayed plant immunity elicitor protein PpAE4 is 0.8-1.5 μM. is spraying the plant immunity elicitor protein PpAE4 onto the surface of a plant; or spraying the recombinant protein of the plant immunity elicitor protein PpAE4 obtained by expressing and purifying a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 1 onto the surface of a plant; the plant immunity elicitor protein PpAE4 is (bl) or (b2): (bl) a protein having an amino acid sequence as shown in SEQ ID NO: 2; (b2) a fusion protein having the same function by linking a protein tag to the N-terminus and / or C-terminus of the protein of (bl); and the disease resistance is the resistance to Phakopsora pachyrhizi; and the plant is soybean. The concentration of the sprayed plant immunity elicitor protein PpAE4 is 0.8-1.5 μM. ​ 2. Use according to claim 1, characterized in that, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 4. Use according to claim 3, characterized in that, ​ 5. A method for increasing disease resistance in plants, characterized by, ​ ​ ​ ​ ​ 6. The method of claim 5, wherein, ​

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