Application of soybean rust fungus elicitor PpE1 protein in inducing plant immune response and / or improving plant resistance

Abstract

The invention provides application of soybean rust fungus elicitor PpE1 protein in inducing plant immune response and / or improving plant resistance, and belongs to the technical field of elicitor protein application. The amino acid sequence of the soybean rust fungus elicitor PpE1 protein is as shown in SEQ ID NO. 2. The invention discovers the application of the soybean rust fungus protein elicitor PpE1 in inducing plant immune response and improving plant resistance for the first time. The PpE1 can cause the hypersensitive reaction of the nicotiana benthamiana and induce active oxygen in the nicotiana benthamiana to burst out; the protein can induce immune response of soybeans, including burst of active oxygen, activation of MAPK and expression of immune genes, and promote resistance of the soybeans to rust fungi; the heterologous expression of the PpE1 can also improve the resistance of the soybean to rust fungi. The invention provides a new way for improving the disease resistance of plants, and has a wide application prospect in agricultural production.

Description

Technical Field

[0001] This invention relates to the field of elicitor protein application technology, and in particular to the application of soybean rust fungus elicitor PpE1 protein in inducing plant immune responses and / or enhancing plant resistance. Background Technology

[0002] Soybeans Glycine max Soybeans are one of the most important oilseed and grain crops. However, soybeans are often threatened by various diseases during production, among which the live nutritional pathogenic fungus *Potamogeton bean rust* is a common culprit. Phakopsora pachyrhizi Soybean rust, caused by [unspecified disease], is a global and devastating disease. It spreads rapidly and causes severe damage, leading to widespread yellowing and shedding of soybean leaves, premature plant death, and yield reductions of up to 30%, resulting in enormous economic losses to the soybean industry.

[0003] Currently, the control of soybean rust in production mainly relies on chemical pesticides. Spraying fungicides to curb the spread of pathogens is the most widely used field management measure. However, this control strategy has problems such as high economic costs, environmental harm, and food safety risks. It also easily leads to drug resistance in soybean rust fungi, creating a vicious cycle. Therefore, developing an efficient, safe, and environmentally friendly alternative control strategy for soybean rust has become a key issue that urgently needs to be addressed in this field.

[0004] In nature, plants have evolved a two-layered immune system to defend against pathogen invasion, primarily relying on two major classes of immune receptors: pattern recognition receptors (PRRs) on the cell surface, which recognize pathogen-associated molecular patterns (PAMPs) and trigger PAMP-induced immune responses (PTI); and intracellular NLR receptors, which recognize pathogen effectors and trigger effector-induced immune responses (ETI). PTI involves the recognition of PAMPs and the activation of basic defense responses, such as the production of reactive oxygen species (ROS), mitogen-activated protein kinases (MAPKs), and the expression of pathogenesis-related protein genes (PRs). Therefore, providing an elicitor that can be recognized by receptors on the plant cell surface to trigger a plant immune response and promote plant disease resistance is a highly promising new approach to plant protection.

[0005] Currently, few elicitors capable of effectively stimulating soybean immune responses have been identified from soybean rust fungi, and their functions and disease-inducing effects still require further investigation. Therefore, cloning and identifying novel and highly efficient immune elicitors from soybean rust fungi, and elucidating their role in soybean rust-resistant immune responses, has significant theoretical value and application prospects for developing elicitor-based biopesticides or breeding new disease-resistant varieties through genetic engineering. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the soybean rust elicitor PpE1 protein in inducing plant immune responses and / or enhancing plant resistance.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of soybean rust elicitor PpE1 protein in inducing plant immune responses and / or enhancing plant resistance, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Preferably, the plants include tobacco and legumes.

[0009] Preferably, the legume includes soybean.

[0010] Preferably, the induction of plant immune response includes inducing the production of reactive oxygen species in plants, activating mitogen-activated protein kinase cascade reactions, or increasing the expression of disease-related protein genes.

[0011] Preferably, the induction of plant immune response includes inducing a reduction in the damage caused by rust to plants; the enhancement of plant resistance includes enhancing the plant's resistance to rust.

[0012] Preferably, the rust disease is caused by the rust fungus *Pseudomonas beanus*.

[0013] This invention provides a method for inducing plant immune responses and / or enhancing plant resistance by introducing the gene encoding the soybean rust elicitor PpE1 protein into a target plant to obtain plants that exhibit immune responses and / or enhanced resistance. Alternatively, soybean rust elicitor PpE1 protein or a composition containing soybean rust elicitor PpE1 protein may be applied to the target plant to obtain plants that exhibit immune response and / or enhanced resistance. The amino acid sequence of the soybean rust exciton PpE1 protein is shown in SEQ ID NO.2.

[0014] Preferably, the nucleotide sequence of the gene encoding the soybean rust elicitor PpE1 protein is shown in SEQ ID NO.1; the plant includes tobacco and legumes.

[0015] Preferably, the induction of plant immune response includes inducing a reduction in the damage caused by rust to plants; the enhancement of plant resistance includes enhancing the plant's resistance to rust.

[0016] This invention provides the application of soybean rust elicitor PpE1 protein in the preparation of pesticides, which are used to induce plant immune responses and / or enhance plant resistance; the amino acid sequence of the soybean rust elicitor PpE1 protein is shown in SEQ ID NO.2.

[0017] Preferably, the plant includes tobacco and legumes; the induction of plant immune response includes inducing a reduction in the damage of rust to the plant; and the enhancement of plant resistance includes enhancing the plant's resistance to rust.

[0018] Preferably, the concentration of the soybean rust elicitor PpE1 protein in the pesticide is 300~700 nmol / L.

[0019] SEQ ID NO.1: ATGTTTGTTTCCCTCAAGGCCATAATTTCGACCACAATCGCTACCCTTGCGGTGATCGGAGTCGTTTCTGGGCAGAATATTACTATAGTCTCTCCACAGAGCGGGCAAGTCATCAGGGCAGGTGATGTGTTAACAGTTGAACTAGCCCAACAATCTACCACATCATCGGTCCAAAACATCGCATTCAGCACCGGTTTCAGTTCAAGGCCCAC AGATACTATTTCCTTGGGCAGACCTTTAGGCGGAACCCAGTTTTTGACTACCAGAGGAAACATAACCTTTCAAATTAATGTGCCCAAAGCAAGTGAATTTATTGACGGGATTTCCAATGTCCCTTACGAATTCAAAGTAGCCCATTACTATTTCCTTGGTGCCACCTCTACTCCCACGATTGATATAGCCAGCGTTCCAGTTAGCGTACAACAATAA

[0020] SEQ ID NO.2: MFVSLKAIISTTIATLAVIGVVSGQNITIVSPQSGQVIRAGDVLTVELAQQSTTSSVQNIAFSTGFSSRPTDTISLLGRPLGTQFLTTRGNITFQINVPKASEFIDGISNVPYEFKVAHYYFLGATSTPTIDIASVPVSVQQ

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover the application of the soybean rust fungus protein elicitor PpE1 in inducing plant immune responses and enhancing plant resistance. PpE1 can induce a hypersensitivity response to *N. benthamiana*, triggering the release of reactive oxygen species (ROS) in *N. benthamiana*; its protein can induce an immune response in soybean, including the release of ROS, activation of MAPKs, and expression of immune genes, and promote soybean resistance to rust fungi; heterologous expression PpE1It can also enhance soybean resistance to rust fungi. This invention demonstrates that the PpE1 protein has an immune inducer function and can be used as a novel biopesticide for the control of plant diseases.

[0022] This invention utilizes the protein elicitor PpE1 to induce plant immune responses and enhance plant disease resistance, demonstrating significant effects and providing a new approach to improving plant disease resistance. It has broad application prospects in agricultural production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The structural map of the GFP-FLAG-202 (35S::FLAG) plant expression vector; Figure 2 The structural map of the pCNG (35S::GFP) plant expression vector; Figure 3 The results show the activation of the immune response to *Nycium benthamianum* by PpE1, where A represents PpE1 expressing the full-length PpE1 and the PpE1 signal peptide without the signal peptide in *Nycium benthamianum*. ΔSP The occurrence of hypersensitivity reactions in the posterior leaflets; B represents PpE1 and PpE1. ΔSP Protein expression in Nicotiana benthamiana, C represents PpE1 and PpE1 ΔSP Subcellular localization results, scale bar 20 μm; Figure 4 The structural map of the pGEX-6P-1 prokaryotic expression vector; Figure 5 The results show the stimulation of the immune response of PpE1 protein to Nicotiana benthamiana. Among them, A shows the purification results of PpE1-GST and GST prokaryotic expression proteins; B shows the cell death induced by PpE1-GST protein in Nicotiana benthamiana; and C shows the results of PpE1-GST protein stimulating the release of reactive oxygen species in Nicotiana benthamiana. Figure 6 The results of PpE1 protein stimulating the immune response in soybean are shown in the figures. A represents the activation of the soybean MAPK response by PpE1-FLAG protein, B represents the induction of reactive oxygen species in soybean by PpE1-FLAG protein, C represents the induction of soybean disease course-related protein gene GmPR1 expression by PpE1-FLAG protein, and D represents the induction of soybean disease course-related protein gene GmPR2 expression by PpE1-FLAG protein. Figure 7The results show the detection of PpE1 transgenic soybean resistance to soybean rust. In this table, A is the expression of PpE1-FLAG transgenic soybean protein identified by Western blot, B is the growth phenotype of PpE1-FLAG transgenic soybean, and C is the resistance of PpE1-FLAG transgenic soybean to rust. The scale bar is 1 cm. Figure 8 To illustrate the resistance of soybean leaves to soybean rust after spraying with PpE1-FLAG protein, the scale bar is 1 cm. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. The soybean rust fungus SS4 strain used in the following embodiments was previously isolated and preserved by researchers in our laboratory. Hao Q et al. A pair of an atypical NLR encoding genes confer Asiansoybean rust resistance in soybean. Nat Commun. 2024. , Chen, H. et al. Genetic analysis and molecular mapping of resistance gene to Phakopsorapachyrhizi in soybean germplasm SX6907. Theor. Appl. Genet. 128, 733–743 (2015). The diseased leaves were obtained by inoculating the Tianlong No. 1 soybean variety with the soybean rust fungus strain SS4. The inoculation method was based on Hao Q et al. A pair of an atypical NLR encoding genes confer Asian soybean rustresistance in soybean. Nat Commun. 2024., specifically: the urediniospores of rust fungus SS4 were washed with sterile water containing 0.1% Tween 20 and the concentration was adjusted to 10. 5 One spore / mL. The rust spore solution was added dropwise or sprayed onto the underside of the leaves of Tianlong No. 1 soybean variety using a pipette. The leaves were cultured for 10-15 days under conditions of 12 / 12h photoperiod, 22-25℃ temperature, and 70% relative humidity to obtain soybean rust-infected leaves.

[0026] Example 1: Acquisition and identification of soybean rust fungus elicitor PpE1

[0027] 1. Total RNA extraction from soybean rust fungi

[0028] Tissue from soybean rust-infected leaves was frozen in liquid nitrogen and ground into powder. The frozen tissue powder (100 mg) was quickly transferred to a 1.5 mL centrifuge tube containing 1 mL of pre-chilled Trizol. The mixture was vigorously vortexed and placed on ice for 10 min. Centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was transferred to a new 1.5 mL centrifuge tube, and 200 μL of chloroform was added. The tube was immediately vigorously vortexed and placed on ice for 10 min. After the mixture separated into layers, it was centrifuged at 12000 rpm for 15 min at 4°C. 500 μL of the supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was vortexed and placed on ice for 10 min. Centrifuged at 12000 rpm for 10 min at 4°C, the supernatant was discarded, and 1 mL of isopropanol was added. Prepare 75% ethanol with DEPC water (ultrapure water treated with diethyl pyrocarbonate and sterilized by high temperature and high pressure), invert and mix well, and resuspend the precipitate. Centrifuge at 12000 r / min for 5 min at 4 °C, and repeat this step once. Place the centrifuge tube empty in the centrifuge for 1 min, then carefully aspirate the residual liquid with a pipette and let it stand at room temperature for 10 min to dry the RNA precipitate. Add 40 μL of DEPC water, and after the precipitate dissolves, store it at -80 °C for later use.

[0029] 2. Gene cloning

[0030] First-strand cDNA was synthesized using the extracted RNA as a template via reverse transcription (Novizan R312). The reaction mixture consisted of 1 μg RNA, 2 μL 5×g DNA wiper Mix, and RNase-free ddH2O to a final volume of 10 μL. The mixture was incubated at 42°C for 2 minutes. Further steps included adding 1 μL primer Oligo(dT)20VN, 2 μL 10×RT Mix, 2 μL HiScript III Enzyme Mix, and 5 μL RNase-free ddH2O to the reaction mixture. The mixture was then incubated in a PCR instrument at 25°C for 5 minutes, followed by incubation at 37°C for 45 minutes and 85°C for 5 seconds to obtain soybean rust cDNA.

[0031] Using cDNA as a template, gene-specific primers with added homologous arms for cloning were used (the primers used for cloning to the 35S::FLAG vector were: PpE1-FLAG-F, PpE1-FLAG-R, PpE1...). ΔSP -FLAG-F, nucleotide sequence as shown in SEQ ID NO.3~5; primers used for cloning into the 35S::GFP vector are PpE1-GFP-F, PpE1 ΔSP-GFP-F and PpE1-GFP-R (nucleotide sequences shown in SEQ ID NO. 6~8) were amplified by PCR to obtain the full-length sequence of the target gene (PpE1-F / PpE1-R) and the PpE1 gene without the signal peptide. ΔSP Sequence (PpE1) ΔSP -F / PpE1-R). Reaction system: Pfu DNA Polymerase 1 μL, Pfu Buffer 10 μL, High Pure dNTPs 2.5 μL, cDNA 2 μL, primers F and R 1 μL each, add ddH2O to bring the total to 50 μL. Reaction conditions: Pre-denaturation 95°C for 2 min; denaturation 94°C for 20 s, annealing 57°C for 20 s, extension 72°C for 15 s, for a total of 35 cycles; final extension 72°C for 5 min, storage at 4°C for 2 min. PCR products were obtained after the PCR reaction. These PCR products contained corresponding homologous arms and could be cloned into the 35S::FLAG and 35S::GFP vectors via homologous recombination. The PCR products were recovered after agarose gel electrophoresis, and some samples were sent for sequencing.

[0032] PpE1-FLAG-F:tttggagaggacacgctcgagATGTTTGTTTCCCTCAAGGCC (SEQ ID NO.3)

[0033] PpE1-FLAG-R: gtctttgtagtccatcccgggTTGTTGTACGCTAACTGGAACGC (SEQ ID NO.4)

[0034] PpE1 ΔSP -FLAG-F:tttggagaggacacgctcgagATGCAGAATATTACTATAGTCTCTCCACA (SEQID NO.5)

[0035] PpE1-GFP-F: agaaggcctactagtggatccATGTTTGTTTCCCTCA (SEQ ID NO. 6)

[0036] PpE1 ΔSP -GFP-F: agaaggcctactagtggatccATGCAGAATATTACTATAGTCTCTCCACAGAGC (SEQ ID NO.7)

[0037] PpE1-GFP-R: accagacccgcctcccccgggTTGTTGTACGCTAACTGGAACGC (SEQ ID NO. 8)

[0038] 3. Carrier Construction

[0039] First use XhoI and Sma I (Thermo Scientific) digested 35S::FLAG plant expression vector ( Figure 1 (When constructing the vector, the target gene was replaced with GFP to obtain a protein fused with the FLAG tag. This protein was then linearized and used...) Bam HI and Sma I (Thermo Scientific) digested 35S::GFP plant expression vector ( Figure 2 Linearize the plasmid DNA. The reaction system consisted of 1.5 μg plasmid DNA, 1 μL of each restriction enzyme, 5 μL of 10×FastDigest Buffer, and ddH2O to bring the total volume to 50 μL. Reaction conditions: incubate at 37℃ for 30 min.

[0040] PpE1 and PpE1 ΔSP The gene fragment was constructed into linearized 35S::FLAG and 35S::GFP vectors using a seamless cloning reaction (Novizan C117). The reaction mixture consisted of 5 μL of 2×CE Mix, 1.5 μL of the gene fragment, and 2.5 μL of the linearized vector, with ddH2O added to a final volume of 10 μL. The reaction mixture was then placed in a PCR chamber for recombination at 50°C for 15 min before transformation.

[0041] During transformation, heat shock transformation of Escherichia coli DH5a was performed as follows: 10 μL of seamless cloning reaction product was added to 50 μL of competent E. coli cells, incubated on ice for 30 min, heat-shocked at 42°C for 60 s, incubated on ice for 2 min, 400 μL of LB medium was added, mixed well, and then restored at 37°C and 200 r / min for 1 h. The mixture was then spread on LB plates containing kanamycin and incubated upside down at 37°C for 12 h until single colonies grew.

[0042] Colony PCR test: White single colonies were picked using a pipette tip for PCR identification. Reaction system: Novizan 2×RapidTaq Master Mix (P222) 10 μL, primer F / R 0.5 μL, H2O 9 μL. Cycling conditions: (Pre-denaturation: 95°C, 2 min; Denaturation: 95°C, 15 s; Annealing: 56°C, 15 s; Extension: 72°C, 15 s; 32 cycles total; Final extension: 72°C, 5 min; Storage: 16°C, 4 min). Single colonies were picked and verified as positive by PCR before being sent to Kexin Technology Co., Ltd. for sequencing. Plasmids with correct sequencing results were extracted for transformation of Agrobacterium GV3101 and EHA105 strains.

[0043] 4. Agrobacterium-mediated transformation

[0044] Add 1 μL of plasmid to 50 μL of GV3101 or EHA105 Agrobacterium competent cells, mix well, transfer to a pre-cooled electroporation cuvette, electroporate on a Bio-Rad in AGR mode, add 400 μL of LB liquid medium, mix well, and revive at 28°C and 200 r / min for 1 h. Spread the mixture on LB plates containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubate upside down at 28°C for 48 h until single colonies grow.

[0045] Colony PCR testing: Single colonies were picked up with a pipette tip for PCR identification. Cycling conditions: (Pre-denaturation: 95°C, 2 min; Denaturation: 95°C, 15 s; Annealing: 56°C, 15 s; Extension: 72°C, 15 s; Final extension: 72°C, 5 min; 32 cycles in total; Storage: 16°C, 4 min). Colonies that tested positive by electrophoresis were stored for transient expression of Nicotiana benthamiana and soybean transformation.

[0046] In this embodiment, PpE1-FLAG and PpE1 were constructed. ΔSP -FLAG, PpE1- GFP, PpE1 ΔSP - Four GFP vectors. The GFP-FLAG vector is a control. In subsequent experiments, PpE1-GFP (GV3101), PpE1... ΔSP -GFP (GV3101) is used for subcellular localization in tobacco. PpE1-FLAG (GV3101), PpE1 ΔSP - FLAG (GV3101) was used for transient expression in *N. benthamiana* to observe cell death response. PpE1-FLAG (GV3101) and the vector control GFP-FLAG (GV3101) were used for transient expression and protein purification in *N. benthamiana*. PpE1-FLAG (EHA105) was used for genetic transformation of soybean to obtain PpE1-FLAG transgenic soybeans.

[0047] Example 2: Study on the role of PpE1 in inducing plant immune responses and enhancing plant resistance

[0048] 1. The stimulation of immune response to Nicotine Benignard by PpE1

[0049] The target vector was transformed into Agrobacterium GV3101 strain via electroporation. Positive single-clone bacterial suspensions were streaked onto LB agar plates containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and incubated at 28°C for 2 days. Single colonies were picked from the plates and transferred to 5 mL of liquid LB containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and cultured at 28°C with shaking at 200 r / min for 20 h. The bacterial cells were collected by centrifugation at 6000 r / min for 5 min, and resuspended in 5 mL of Agrobacterium injection buffer containing 10 mM MgCl2, 10 mM MES, and 200 mM AS. The OD600 was adjusted to 0.5, and the suspension was allowed to stand at room temperature for 2 h. The bacterial suspension was then injected into the lower epidermis of tobacco leaves using a 1 mL syringe (without the needle) to obtain Nicotiana benthamiana transiently expressing the corresponding vector.

[0050] PpE1 and PpE1 of the corresponding carrier, nicotine Benedictine, were expressed transiently. ΔSP Protein expression was detected using the following method: Five leaf discs with a diameter of 6 mm were ground using a punch, and 100 μL of SDS Load Buffer (100 mM Tris-HCl pH=6.8, 8% SDS, 40% glycerol, 2 mM DTT, 0.004% bromophenol blue) was added. After vigorous shaking, the mixture was denatured at 95-100℃ for 5-10 min, centrifuged at high speed for 5 min, and 20 μL of supernatant was loaded onto the sample for Western blot detection using FLAG antibody.

[0051] PpE1 and PpE1 were observed using fluorescence methods. ΔSP Subcellular localization was performed using the following method: PpE1-GFP and PpE1 were expressed transiently. ΔSP - GFP and GFP control strain GV3101 were injected into leaves of *Nicotiana benthamiana*. Two to three days post-inoculation, GFP fluorescence signals were observed using a confocal microscope (Zeiss, LSM980). GFP was detected after excitation with a 488 nm laser, with the emission light collection range being 500 to 550 nm. Laser intensity was set between 1.0 and 3.0, and gain between 500 and 550.

[0052] Agrobacterium-mediated transient expression of both full-length (secretory extracellular) and PpE1 lacking the signal peptide in Nicotiana benthamiana. ΔSP(It cannot be secreted extracellularly). Results showed that PpE1 can induce Benedict's tobacco hypersensitivity, while PpE1... ΔSP It cannot cause hypersensitivity reactions to Benedict's tobacco. Figure 3 (A, B). Fluorescence observation revealed that PpE1 ΔSP -GFP is localized similarly to the empty GFP vector, while PpE1-GFP is localized to the apoplast ( Figure 3 (C). This indicates that PpE1 is recognized by receptors on plant cell membranes and elicits an immune response, demonstrating its function as an immune elicitor.

[0053] Subsequently, the PpE1 CDS sequence without the signal peptide was constructed into the pGEX-6P-1 prokaryotic expression vector ( Figure 4 The vector's restriction enzyme sites are BamHI and EcoRI. The primers used to amplify PpE1 are PpE1-F (SEQ ID NO. 9) and PpE1-R (SEQ ID NO. 10). The vector construction and verification method is the same as in Example 1. The constructed vector was transformed into competent cells of BL21(DE3) Escherichia coli. The strain containing PpE1 (PpE1-GST) or the control (pGEX-6P-1 / GST) was added to 200 mL of LB liquid medium and shaken. The culture was incubated at 37℃ for 2-3 h until the OD600 value was 0.5. Then, IPTG (final IPTG concentration of 0.25 mM) was added to 200 mL of LB medium, and the culture was induced at 25-28℃ and 160 rpm for 10 h. The cells were collected by centrifugation at 5000 rpm and 4℃ and resuspended in PBS buffer. The E. coli were then disrupted by sonication (Xinzhi JY99-IIDN, 6 mm amplitude transformer, output power 30%). After ultrasonic disruption, the supernatant was collected by centrifugation at 4°C and 12,000 rpm for 15 min. PpE1-GST or GST protein was enriched with Glutathione Resin (GenScriptCat.No.L00206) and eluted with PBS buffer containing 10 mM reduced glutathione to obtain PpE1-GST or GST protein.

[0054] PpE1-F: ttccaggggcccctgggatccCAGAATATTACTATAGTCTCTCCACAGAGC (SEQ IDNO.9)

[0055] PpE1-R: ctcgagtcgacccgggaattcTTGTTGTACGCTAACTGGAACGC (SEQ ID NO. 10)

[0056] Protein purification results are as follows Figure 5As shown in Figure A, when PpE1-GST was injected into tobacco leaves (at a concentration of 500 nM, in an area the size of a coin), it was found that PpE1-GST could induce cell death in tobacco leaves. Figure 5 (B), and can be used as a PAMP-induced ROS burst in Benedictine smoke ( Figure 5 (C)

[0057] The ROS detection method is as follows: Take leaf discs with a diameter of 6 mm from 4-5 week old tobacco leaves by punching holes. Cut each leaf disc into 4 narrow strips using a scalpel. Place the leaf discs into 96-well plates, add 100 μL of ddH2O to each well, and allow to recover overnight. Remove the ddH2O, and add 100 μL of reaction mixture, including 50 μM Luminol, 10 μg / mL peroxidase, and 500 nM PpE1-GST protein or GST control (n=12). Place the samples in a microplate reader and start the measurement immediately, measuring every 1.5 minutes, repeating 35 times.

[0058] PpE1-FLAG and GFP-FLAG proteins were transiently expressed in Nicotiana benthamiana (using the same method as above), and purified by FLAG affinity gel extraction. The specific purification method was as follows: Samples were collected 48 h after injection into tobacco leaves. Leaf veins were removed, and the leaves were rapidly ground into powder in liquid nitrogen. Approximately 5 g of powder was transferred to a centrifuge tube containing 10 mL of pre-cooled RIPA lysis buffer (weak) (Beyotime, P0013D). PMSF (another protease inhibitor) was added to achieve a final concentration of 1 mM. The powder was vortexed to ensure uniform suspension and incubated on ice for 30 min. The mixture was then centrifuged at 12000 r / min for 15 min at 4°C. The supernatant was transferred to a new centrifuge tube, and the centrifugation was repeated once. The supernatant was then filtered again through a 0.22 μm bacterial filter. 500 μL of FLAG affinity gel (GenScript, L00425) was added to the filtrate, and the mixture was incubated at 4°C with gentle shaking for 4 h. Centrifuge at 1000 r / min for 1 min, remove the supernatant, collect the beads, add 1 mL of RIPA lysis buffer (weak) to wash, repeat 4 times, add 3×FLAG peptide to elute and obtain the protein.

[0059] 2. The stimulation of soybean immune response by PpE1

[0060] Leaf discs with a diameter of 6 mm were collected from the leaves of Tianlong No. 1 soybeans grown for 4 weeks and treated with a 500 nM protein solution. The ROS detection method was the same as above. For MAPK detection: four 6 mm diameter leaf discs were placed in a cell culture dish and 1 ml of H2O was added for overnight recovery. The H2O was removed from the culture dish, and 500 μL of a 500 nM PpE1-GST or GST protein solution was added. Leaf discs were collected at 0 min and 15 min after treatment. After being ground in liquid nitrogen, the protein was extracted using the previous method, and Western blot analysis was performed using pERK1 / 2 antibody (Cell Signaling Technology, 9101) (method as before) to detect MAPK activation. PR1 and PR2 expression level detection: Four leaf discs with a diameter of 6 mm were placed in a cell culture dish and 1 mL of H2O was added for overnight recovery. The H2O was removed from the culture dish, and 500 μL of 500 nM PpE1-GST or GST protein solution was added. The leaf discs were collected after 12 h of treatment. RNA was extracted using the Novizan RNA Extraction Kit (RC411). The expression levels of PR1 and PR2 were detected by qRT-PCR. The primers used for detection are shown in SEQ ID NO.11~SEQ ID NO.16. The reaction system was iTaq. TM 5 μL of Universal SYBR® Green Supermix (Bio Rad), 0.5 μL each of primers F and R, 2 μL of cDNA, and 2 μL of H2O. Reaction conditions: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 40 cycles; 65℃~95℃, increasing by 0.5℃ per cycle for 5 s. The reaction was performed on a Bio Rad CFX 96 real-time PCR instrument. After amplification, the amplification result was obtained by... -ΔΔCT The method calculates the relative expression level of genes.

[0061] GmPR1-F: ATGGGGTCGGGGATTAAGG (SEQ ID NO.11)

[0062] GmPR1-R: CAGCGTTGTGTGCATTAACAAA (SEQ ID NO.12)

[0063] GmPR2-F: GTCTCCTTCGGTGGTAGTG (SEQ ID NO.13)

[0064] GmPR2-R:ACCCTCCTCCTGCTTTCTC (SEQ ID NO.14)

[0065] Internal control primer Gmubiquitin 3-F: GTGTAATGTTGGATGTGTTCCC (SEQ ID NO.15)

[0066] Internal control primer Gmubiquitin 3-R: ACACAATTGAGTTCAACACAAACCG (SEQ ID NO.16)

[0067] The results showed that PpE1-FLAG could stimulate the MAPK cascade, ROS bursting, and expression of pathogenesis-related proteins GmPR1 and GmPR2 in soybean. Figure 6 (A~D). This indicates that PpE1 can activate the disease resistance response in soybeans.

[0068] 3. Control of soybean diseases using PpE1

[0069] Construction of transgenic soybean: Using the soybean cultivar Tianlong No. 1 as the genetic transformation recipient, the Agrobacterium-mediated soybean cotyledonary node genetic transformation method (Paz MM, Martinez JC, Kalvig AB, Fonger TM, Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep, 2006, 25(3): 206-213) was used to transform soybeans with recombinant Agrobacterium EHA105 / 35S:: PpE1-FLAG. The transformed plants were screened by applying 100 mg / L glufosinate herbicide to the leaves, and T0 generation soybean transformation materials were obtained. The protein expression of PpE1-FLAG was detected by western blot after extracting soybean leaf protein according to the previous method. After being identified as positive transformation plants, they were planted in a greenhouse for further generation under the conditions of 25℃, 12h light / 12h dark. Phenotypic analysis was performed on OE6 and OE10 families.

[0070] Resistance testing for soybean rust fungus: Tianlong No. 1 (TL-1) soybean variety, PpE1 transgenic soybeans OE6 and OE10 (TL-1 recipient), were cultivated in a greenhouse at 25 °C with a 16-hour photoperiod. Leaves of OE6, OE10, and Tianlong No. 1 soybeans were transferred to petri dishes, with two layers of absorbent paper and filter paper underneath for moist cultivation. Uredinium spores of soybean rust fungus strain SS4 were washed with sterile water containing 0.1% Tween 20 and the concentration was adjusted to 10. 51 spore / mL. The rust spore solution was sprayed onto the underside of soybean leaves, with each leaf sprayed 3 times (0.5 mL of spore solution). The leaves were cultured for 10 to 15 days under a photoperiod of 12 / 12h, a temperature of 22-25℃, and a relative humidity of 70%. The number of rust spore masses in the leaves of transgenic soybeans and control soybeans was compared to assess their resistance to rust.

[0071] The expression of PpE1 protein was examined by Western blot. Figure 7 (A). Inoculation of the PpE1 transgenic soybean families OE6 and OE10 with soybean rust fungus revealed significantly enhanced resistance to soybean rust fungus. Figure 7 (B and C). Because PpE1 can trigger an immune response in soybeans, the continuous and stable expression of PpE1 in soybeans will continue to induce an immune response, resulting in a state of over-immunity in transgenic soybeans and inhibited growth.

[0072] Tianlong No. 1 (TL-1) soybean leaves were sprayed with GFP-FLAG and PpE1-FLAG proteins purified from tobacco at a concentration of 500 nM. One to two hours after spraying, once the leaf surface showed no obvious moisture, the leaves were inoculated using the aforementioned method at a concentration of 10 nM. 5 Spores / mL of soybean rust urediniospores; statistical analysis after 10 days of inoculation revealed that soybean leaves treated with PpE1 protein showed significantly enhanced resistance to rust fungus. Figure 8 This indicates that PpE1 protein can be used as a biopesticide to control soybean rust. Spraying plants with PpE1 protein to induce resistance does not affect plant growth and may even enhance disease resistance.

[0073] In summary, this invention is the first to discover the role of the soybean rust protein elicitor PpE1 in inducing plant immune responses and enhancing soybean rust resistance, providing a new approach to improving plant disease resistance.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of soybean rust elicitor PpE1 protein in inducing plant immune responses and / or enhancing plant resistance, characterized in that, The amino acid sequence of the soybean rust exciton PpE1 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The plants mentioned include tobacco and legumes.

3. The application according to claim 1, characterized in that, The induced plant immune response includes inducing the production of reactive oxygen species, activating mitogen-activated protein kinase cascades, or increasing the expression of disease-related protein genes.

4. The application according to claim 1, characterized in that, The induction of plant immune response includes inducing a reduction in the damage caused by rust to plants; the enhancement of plant resistance includes enhancing the plant's resistance to rust.

5. The application according to claim 4, characterized in that, The rust disease is caused by the rust fungus *Pseudomonas beanus*.

6. A method for inducing plant immune responses and / or enhancing plant resistance, characterized in that, The gene encoding the soybean rust elicitor PpE1 protein was introduced into the target plant to obtain plants that exhibited an immune response and / or increased resistance. Alternatively, soybean rust elicitor PpE1 protein or a composition containing soybean rust elicitor PpE1 protein may be applied to the target plant to obtain plants that exhibit immune response and / or enhanced resistance. The amino acid sequence of the soybean rust exciton PpE1 protein is shown in SEQ ID NO.

2.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the gene encoding the soybean rust elicitor PpE1 protein is shown in SEQ ID NO.1; the plant includes tobacco and legumes.

8. The method according to claim 6, characterized in that, The induction of plant immune response includes inducing a reduction in the damage caused by rust to plants; the enhancement of plant resistance includes enhancing the plant's resistance to rust.

9. The application of soybean rust fungus elicitor PpE1 protein in pesticide preparation, characterized in that, The pesticide is used to induce plant immune responses and / or enhance plant resistance; the amino acid sequence of the soybean rust elicitor PpE1 protein is shown in SEQ ID NO.

2.

10. The application according to claim 9, characterized in that, The plants include tobacco and legumes; the induction of plant immune response includes inducing a reduction in the damage of rust to the plants; the enhancement of plant resistance includes enhancing the plants' resistance to rust.

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