Plant immunity-inducing protein cche1 and use thereof

Abstract

The present invention belongs to the field of biotechnology. Disclosed are a plant immunity-inducing protein CcHE1 and a use thereof. The disclosed plant immunity-inducing protein CcHE1 secreted by a plant pathogenic fungus has an amino acid sequence shown as SEQ ID NO.2, and a nucleotide sequence of the gene encoding CcHE1 shown as SEQ ID NO.1. The present invention provides a novel plant immunity-inducing protein CcHE1, capable of inducing plant resistance and immune defense responses, significantly enhancing plant disease resistance, effective at low concentrations, and exhibiting remarkable disease-resistance effects. CcHE1 activates the plant's own immune system, providing a new approach to improving plant resistance, and has broad application prospects in areas such as green control of forest diseases.

Description

Plant immune elicitor protein CcHE1 and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a plant immune elicitor protein CcHE1 and application thereof. BACKGROUND

[0002] Poplar is an important afforestation tree species in China, with strong adaptability, easy growth, and poor tolerance, etc. It can show good rapid growth and resistance in hilly and mountainous areas, and become the main tree species of the "three north" shelterbelt system in China. With the large-scale popularization and planting of poplar and poor site environment conditions, poplar diseases are becoming more and more serious. Poplar decay caused by Cytospora chrysosperma is one of the most serious diseases, which causes branches to wither and dry, and even causes tree death and forest destruction. Poplar decay is a host dominant disease, and the strength of host resistance affects the severity of the disease. At present, the prevention and control of the disease mainly adopts passive chemical and physical control, and the input-output effect is poor. With the major strategic demand of the state for green control of plant diseases, it is urgent to develop new green products for the prevention and control of forest diseases.

[0003] During the process of harmful organisms damaging host plants, a large amount of effectors will be secreted, some of which can induce the immune response of plants, which are called elicitors. After the elicitors are recognized by the receptor molecules on the surface of the cell membrane or in the cells of the host plant, a series of signal transduction pathways are triggered, thereby stimulating the defense system of the plant and inducing the defense response of the plant, so that the plant eventually produces systemic resistance and reduces the damage of the harmful organisms. The development of potential elicitors into plant immune inducers for the green control of plant diseases will help to improve the long-standing problems of ecological system destruction, environmental pollution and agricultural and forestry food safety in China, and meet the major strategic demand of the zero growth target of chemical pesticide use. However, there is no related report on the plant immune elicitor protein CcHE1 secreted by Cytospora chrysosperma (Pers.) Fr. in the prior art. SUMMARY

[0004] In order to solve the above-mentioned deficiencies in the prior art, the first object of the present application is to provide a plant immune elicitor protein CcHE1 secreted by a plant pathogenic fungus, the amino acid sequence of which is shown as SEQ ID NO. 2.

[0005] The protein CcHE1 is a newly identified secreted protein from the strain of Cytospora chrysosperma (Pers.) Fr.

[0006] A second object of the present application is to provide a gene encoding the plant immunity elicitor protein CcHE1, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] A third object of the present application is to provide a recombinant vector, an expression cassette or a recombinant bacterium comprising the gene.

[0008] The recombinant vector can be a prokaryotic expression vector comprising the gene encoding CcHE1 from which the signal peptide has been removed, and specifically can be a prokaryotic expression vector comprising the gene encoding CcHE1 from which the signal peptide has been removed in pET-28a. In the specific implementation process, the recombinant vector can be used for expression in E. coli BL21 (DE3) to obtain a fusion expression protein (His-CcHE1) with a molecular weight of about 13.5 kDa, which can induce resistance response in plants such as poplar, tobacco and chestnut, improve the immunity of the plants, and reduce the damage degree of plant diseases caused by Botrytis cinerea, Cytospora chrysosperma, Colletotrichum gloeosporioides, Botryosphaeria dothidea and Cryphonectria parasitica.

[0009] A fourth object of the present application is to provide the use of the plant immunity elicitor protein CcHE1, the gene, the recombinant vector, the expression cassette or the recombinant bacterium in inducing plant defense response or improving plant disease resistance.

[0010] Further, the plant is poplar, tobacco or chestnut.

[0011] Further, the disease resistance is against diseases caused by Botrytis cinerea, Cytospora chrysosperma, Colletotrichum gloeosporioides, Botryosphaeria dothidea and Cryphonectria parasitica.

[0012] A fifth object of the present application is to provide a method for preventing and treating plant diseases, which comprises treating plants with the plant immunity elicitor protein CcHE1.

[0013] Further, the specific treatment method comprises spraying or smearing the plant immunity elicitor protein CcHE1 on the surface of the plants or injecting the plant immunity elicitor protein CcHE1 into the leaves of the plants.

[0014] Further, the plant is poplar, tobacco or chestnut.

[0015] Further, the plant disease is a disease caused by Botrytis cinerea, Cytospora chrysosperma, Colletotrichum gloeosporioides, Botryosphaeria dothidea and Cryphonectria parasitica.

[0016] The embodiment exemplarily verifies that CcHE1 improves the resistance of plants to Botrytis cinerea, Ceratocystis rivariabilis, G. cingulata, Eutypa lata and Cryphonectria parasitica. Since CcHE1 can cause the defense response of plants, induce active oxygen burst, callose deposition, improve the immunity of plants, and further improve the expression of disease resistance genes, a person skilled in the art can reasonably expect that the CcHE1 protein, the gene encoding the CcHE1 protein, the recombinant vector, the expression cassette or the recombinant bacteria containing the gene can improve the resistance of plants to other common plant pathogenic fungi.

[0017] The present application has the following beneficial effects:

[0018] The present application provides a new plant immune elicitor protein CcHE1, which can induce the resistance and immune defense response of plants, significantly improve the disease resistance of plants, and be applied to the prevention and control of plant diseases as a plant immune inducer. Moreover, the protein has a low use concentration and does not cause plant necrosis but can induce the immune defense response of plants, thereby improving the disease resistance of plants to Botrytis cinerea, Ceratocystis rivariabilis, G. cingulata, Eutypa lata and Cryphonectria parasitica. CcHE1 activates the immune system of plants, providing a new way to improve plant resistance, and has a wide application prospect in the green control of forest diseases and the like. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 is a Western blot detection diagram of the plant immune elicitor protein CcHE1 obtained after expression and purification by a prokaryotic expression system, wherein the antibody used is anti-His; and the left side is a coomassie brilliant blue staining, and the right side is a protein immunoblot.

[0020] Fig. 2 is a result diagram of trypan blue staining, active oxygen burst and callose deposition of Nicotiana benthamiana treated with 10 μM, 8 μM and 5 μM plant immune elicitor protein CcHE1.

[0021] Fig. 3 is an expression result of tobacco disease resistance related genes after tobacco leaves are injected with 5 μM plant immune elicitor protein CcHE1.

[0022] Fig. 4 is a disease symptom diagram of Botrytis cinerea inoculated on Nicotiana benthamiana treated with 5 μM plant immune elicitor protein CcHE1.

[0023] Fig. 5 is a result diagram of active oxygen burst and callose deposition of poplar leaves treated with 5 μM plant immune elicitor protein CcHE1.

[0024] Fig. 6 is an expression result of poplar disease resistance related genes after poplar leaves are treated with 5 μM plant immune elicitor protein CcHE1.

[0025] Figure 7 is a diagram of the disease symptoms of poplar leaves inoculated with Colletotrichum gloeosporioides after treatment with 5 μM plant immune elicitor CcHE1.

[0026] Figure 8 is a diagram of the disease symptoms of poplar branches inoculated with Magnaporthe grisea after treatment with 5 μM plant immune elicitor CcHE1.

[0027] Figure 9 is a diagram of the disease symptoms of poplar branches inoculated with Botryosphaeria dothidea after treatment with 5 μM plant immune elicitor CcHE1.

[0028] Figure 10 is a diagram of the disease symptoms of chestnut branches inoculated with Cryphonectria parasitica after treatment with 5 μM plant immune elicitor CcHE1.

[0029] Figure 11 is a diagram of the disease symptoms of poplar branches inoculated with Botryosphaeria dothidea 36 h after treatment with 5 μM plant immune elicitor CcHE1.

[0030] Figure 12 is a diagram of the results of 5 μM plant immune elicitor CcHE1 having no growth inhibitory effect on Botrytis cinerea, Magnaporthe grisea, Colletotrichum gloeosporioides, Botryosphaeria dothidea, and Cryphonectria parasitica. DETAILED DESCRIPTION

[0031] The following examples are intended to be illustrative only and are not intended to limit the scope of the present application. Unless otherwise indicated, the conditions in the examples were carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise indicated, the reagents or instruments used were conventional products that can be obtained commercially.

[0032] Example 1: Cloning of the plant immune elicitor CcHE1-encoding gene

[0033] The cloning of the plant immune elicitor CcHE1-encoding gene was carried out as follows:

[0034] (1) Total RNA extraction: The total RNA was extracted from the mycelium of Magnaporthe grisea cultured in liquid medium using the RNA extraction kit from TIANGEN according to the manufacturer's instructions, and the RNA content and quality were detected using Nanodrop.

[0035] (2) Reverse transcription to generate the first strand: 2 μg of RNA was used as a template, and cDNA synthesis was carried out according to the manufacturer's instructions for the Hifair reverse transcriptase kit from YEASEN, and the volume was adjusted to 40 μL. An appropriate amount of the reverse transcription product was used for the subsequent gene cloning PCR.

[0036] (3) Using the synthesized cDNA as a template and CcHE1-5F and CcHE1-3F as primers, the full-length CcHE1-encoding gene was amplified by ordinary PCR:

[0037] The sequences of the PCR primers are as follows:

[0038] CcHE1-5F: 5'-ATGCAGATCACCACTACCAT-3' (SEQ ID NO. 3);

[0039] CcHE1-3F: 5'-GTACCCCGGGACAGCATCAT-3' (SEQ ID NO. 4).

[0040] 50 μL reaction system was: Sangon Biotech 2xSanTaq PCR Mix enzyme 25 μL, template cDNA 1 μL, CcHE1-5F 2 μL, CcHE1-3F 2 μL, water to 50 μL; PCR amplification procedure was 94°C pre-denaturation 5 min, 94°C denaturation 30 s, 55°C annealing 30 s, 72°C extension 15 s, cycle 35 times, 72°C extension 10 min.

[0041] (4) The obtained band was detected by agarose gel electrophoresis to determine whether it was the target band, and the PCR product of CcHE1 coding gene was further recovered by cutting the gel, that is, the plant immune elicitor protein CcHE1 coding gene was obtained.

[0042] The full-length nucleotide sequence of Cytospora chrysosperma (Pers.) Fr. CcHE1 coding gene is 396 bp long, and the specific sequence is as follows:

[0043] The full-length protein sequence of Cytospora chrysosperma (Pers.) Fr. CcHE1 is 131 aa long, and the specific sequence is as follows:

[0044] Example 2: Prokaryotic expression and purification of plant immune elicitor protein CcHE1

[0045] The prokaryotic expression and purification process of plant immune elicitor protein CcHE1 is as follows:

[0046] (1) Construction of prokaryotic expression vector

[0047] The specific primers of plant immune elicitor protein CcHE1 coding gene were designed, and the primer sequences were as follows:

[0048] CcHE1-P5F: 5'-CATGCCATGGATGGCTCCCTATGAGTGCACCTT-3' (SEQ ID NO. 5);

[0049] CcHE1-P3F: 5'-CCGCTCGAGGTACCCCGGGACAGCATCAT-3' (SEQ ID NO. 6).

[0050] PCR reaction system: Sangon Biotech 2xSanTaq PCR Mix enzyme 25 μL, template CcHE1 1 μL, CcHE1-P5F 2 μL, CcHE1-P3F 2 μL, water to 50 μL; PCR amplification program: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 15 s, 35 cycles, 72°C extension for 10 min.

[0051] The obtained band was detected by agarose gel electrophoresis to determine whether it was the target band, and the CcHE1 coding gene PCR product carrying NcoI and XhoI (ABclonal) enzyme cutting sites was further recovered by cutting the gel.

[0052] The above gel-recovered DNA and pET-28a plasmid were double-digested, and the 50 μL reaction system was: template DNA / pET-28a 15 μL, 10xK Buffer 5 μL, BSA 5 μL, NcoI 1 μL, XhoI 1 μL, water to 50 μL, and incubated in a 37°C incubator overnight for enzyme digestion, and recovered by using a general DNA product purification kit (TIANGEN). Subsequently, T4 DNA Ligase (ABclonal) was used for ligation, and the 10 μL reaction system was: T4 DNA Ligase 1 μL, 10xT4 DNA Ligase Reaction Buffer 2 μL, enzyme-digested pET-28a 3 μL, enzyme-digested CcHE1 4 μL, and incubated in a 16°C metal bath overnight for ligation. The above ligation product was transformed into E. coli competent cells BL21 (DE3), and LB (containing Kanamycin) plates were coated, and after 12 h of inverted culture at 37°C, colony PCR verification was performed, three positive clones were picked and shaken, plasmid extraction was performed according to the plasmid extraction kit operation (TIANGEN), and sequencing was performed by Beijing Qikong Biotechnology Co., Ltd., sequence alignment was performed on the MUSCLE multiple sequence alignment website, and vector construction was completed.

[0053] (2) Prokaryotic expression of CcHE1

[0054] The recombinant plasmid containing bacteria monoclone was picked into 50 mL of liquid LB medium containing 100 μg / mL Kanamycin, 37°C, 220 rpm, and cultured overnight for 16 h; the overnight cultured E. coli liquid was inoculated into 250 mL of liquid LB medium containing 100 μg / mL Kanamycin at a concentration of 1:50, 37°C, 220 rpm, and expanded to OD 600 = 0.04; 37°C incubation for 2-3 h, and OD 600 = 0.5-0.6; IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the cultured E. coli liquid to a final concentration of 0.2 mM, 20°C, 160 rpm, and cultured for 18 h to induce protein expression; the induced expression of E. coli liquid was centrifuged at 4°C, 4000 rpm, for 15 min, and the supernatant was discarded; the bacteria were lysed using a His-tag protein purification kit (denaturant type) from Beyotime Company, and before lysing the bacteria, an appropriate amount of protease inhibitor cocktail was added to the lysis solution; for fresh precipitate, the lysis solution was added at a ratio of 4 mL non-denaturing lysis solution per gram of wet precipitate, and the bacteria were resuspended thoroughly; lysozyme was added to a final concentration of 1 mg / mL and mixed well, and placed on ice for 30 min; the bacteria were lysed on ice, with an ultrasonic power of 200-300 W, 10 s each time, with an interval of 10 s, for a total of 20-30 min; centrifuged at 4°C, 12000 rpm for 30 min, and the bacterial lysis solution supernatant was collected and placed on ice; 1 mL of mixed 50% BeyoGold TM His-tag Purification Resin (denaturant type) was added to a 2 mL EP tube, centrifuged at 4°C, 3000 rpm for 30 s, and the supernatant was discarded; 0.5 mL of non-denaturing lysis solution was added to the gel and mixed well to equilibrate the gel, centrifuged at 4°C, 3000 rpm for 30 s, and the liquid was discarded. Equilibrium was repeated a total of 3 times, the liquid was discarded, and transferred to a 50 mL centrifuge tube; about 4 mL of bacterial lysis solution supernatant was added, and slowly shaken on a side-to-side shaker or a horizontal shaker at 4°C for 2 h; the mixture of lysis solution and BeyoGold TM His-tag Purification Resin (denaturant type) was loaded into the affinity chromatography column empty tube provided by the kit, the cover at the bottom of the purification column was opened, and the liquid in the column was allowed to flow out under the action of gravity, and the column was washed with 1 mL of 10 mM, 20 mM imidazole buffer in turn, followed by the addition of 500 μL of 500 mM imidazole buffer to elute the target protein, which was transferred to a 2 mL EP tube, and then 500 μL of 500 mM imidazole buffer was added to elute the target protein, which was then transferred to the same EP tube; a precast gel from Changzhou Boyi Biological Technology Co., Ltd. was used for gel electrophoresis to test the protein expression in the above eluate, and subsequent protein purification was performed.

[0055] (3) Purification of CcHE1 protein

[0056] The above eluate was placed in a 15 mL ultrafiltration centrifuge tube, washed with 10 mL of 1xPBS at 4°C, 4500 rpm for 30 min, 3 times; when the total protein solution was concentrated to 1 mL, it was the purified His-tagged CcHE1 protein sample; then the protein concentration was detected using a BCA kit (Solarbio), and electrophoresis, Coomassie blue staining and Western blot detection were performed to detect the purified target protein, and the results are shown in Figure 1.

[0057] As can be seen from Figure 1, electrophoresis detection obtained a C-terminal His-tagged CcHE1 recombinant protein with a molecular weight of about 13.5 kDa.

[0058] Example 3: Defense response induced by plant immune elicitor protein CcHE1 in tobacco

[0059] The plant immune elicitor protein CcHE1 obtained in Example 2 was used in tobacco to induce a defense response, and the specific process was as follows:

[0060] (1) 5 μM CcHE1 does not cause tobacco necrosis but can induce tobacco active oxygen burst and callose deposition

[0061] After determining the concentration of CcHE1 protein, its concentration was adjusted to 10 μM, 8 μM and 5 μM. The middle leaves of tobacco growing to 5-leaf stage were selected, a needle hole was first made at different positions of the tobacco leaves with a needle, and then 1 mL of a syringe without a needle was used to inject different concentrations of CcHE1 protein into the leaves from the back of the leaves. At the same time, PBS was used as a control, and the samples were collected after 48 h of injection. Dead cells of tobacco were stained with trypan blue (Solarbio), and the tobacco was immersed in a fixing liquid (acetic acid: ethanol = 3:1) for 48 h, then the fixing liquid was discarded, and the leaves were washed twice with distilled water, immersed in a staining liquid (0.67 g trypan blue, 100 mL phenol, 100 mL lactic acid, 100 mL glycerol, 100 mL distilled water, 600 mL ethanol) for 12 h at room temperature, and then the leaves were taken out of the staining liquid and immersed in a decolorizing liquid (2 g / mL chloral hydrate) for 48 h, after which the tobacco necrosis was observed; for active oxygen detection, the tobacco leaves injected for 48 h were immersed in a 3,3'-diaminobenzidine (DAB) solution (Solarbio) for 12 h, and then decolorized with 95% ethanol and observed; for callose observation, the tobacco leaves injected for 48 h were immersed in a decolorizing liquid (acetic acid: ethanol = 1:6) for 12 h, and then dyed with aniline blue solution for 12 h, and the deposition of callose was observed using a fluorescence universal microscope (BX61). The above experiments were repeated three times, and the results are shown in Figure 2.

[0062] As shown in FIG. 2, 5 μM CcHE1 did not cause tobacco necrosis but induced tobacco active oxygen burst and callose deposition.

[0063] (2) 5 μM CcHE1 induced the expression level of tobacco resistance-related genes to significantly increase

[0064] After injection of tobacco for 12 h, samples were collected for detection of the expression level of resistance-related genes. RNA extraction was performed using a RNA extraction kit from TIANGEN according to the instructions, and the RNA content and quality were detected using a Nanodrop.

[0065] Reverse transcription to generate first strand: 2 μg of RNA was taken as a template, cDNA synthesis was performed according to the instructions for using Hifair reverse transcriptase from YEASEN, and the volume was adjusted to 40 μL. The reverse transcription product was diluted 4 times for real-time quantitative PCR reaction to detect the expression level of induced resistance-related genes NbHSR203J (the sequence is shown in SEQ ID NO. 27), NbHIN1 (the sequence is shown in SEQ ID NO. 28), NbWRKY8 (the sequence is shown in SEQ ID NO. 29), NbAcre31 (the sequence is shown in SEQ ID NO. 30), NbWRKY7 (the sequence is shown in SEQ ID NO. 31), and NbPR1 (the sequence is shown in SEQ ID NO. 32).

[0066] The primers used in the real-time fluorescent quantitative PCR reaction are as follows:

[0067] NbActin upstream primer NbActin-5F:

[0068] 5'-TGGTCGTACCACCGGTATTGTGTT-3' (SEQ ID NO. 7);

[0069] NbActin downstream primer NbActin-3F:

[0070] 5'-TCACTTGCCCATCAGGAAGCTCAT-3' (SEQ ID NO. 8);

[0071] NbHSR203J upstream primer NbHSR203J-5F:

[0072] 5'-ACGCAGATTTCAACCGAGTAT-3' (SEQ ID NO. 9);

[0073] NbHSR203J downstream primer NbHSR203J-3F:

[0074] 5'-GCCAGTCGCATTGGAGATAA-3' (SEQ ID NO. 10);

[0075] NbHIN1 upstream primer NbHIN1-5F:

[0076] 5'-CCAACTTGAACGGAGCCTATTA-3' (SEQ ID NO. 11);

[0077] NbHIN1 downstream primer NbHIN1-3F:

[0078] 5'-AGGCATCCAAAGAGACAACTAC-3' (SEQ ID NO. 12);

[0079] NbWRKY7 upstream primer NbWRKY7-5F:

[0080] 5'-CACAAGGGTACAAACAACACAG-3' (SEQ ID NO. 13);

[0081] NbWRKY7 downstream primer NbWRKY7-3F:

[0082] 5'-GGTTGCATTTGGTTCATGTAAG-3' (SEQ ID NO. 14);

[0083] NbWRKY8 upstream primer NbWRKY8-5F:

[0084] 5'-AACAATGGTGCCAATAATGC-3' (SEQ ID NO. 15);

[0085] NbWRKY8 downstream primer NbWRKY8-3F:

[0086] 5'-TGCATATCCTGAGAAACCATT-3' (SEQ ID NO. 16);

[0087] NbAcre31 upstream primer NbAcre31-5F:

[0088] 5'-AATTCGGCCATCGTGATCTTGGTC-3' (SEQ ID NO. 17);

[0089] NbAcre31 downstream primer NbAcre31-3F:

[0090] 5'-GAGAAACTGGGATTGCCTGAAGGA-3' (SEQ ID NO. 18);

[0091] NbPR1 upstream primer NbPR1-5F:

[0092] 5'-CCGCCTTCCCTCAACTCAAC-3' (SEQ ID NO. 19);

[0093] NbPR1 downstream primer NbPR1-3F:

[0094] 5'-GCACAACCAAGACGTACTGAG-3' (SEQ ID NO. 20).

[0095] The PCR reaction system comprises 0.5 μL of cDNA, 10 μL of qPCR SYBR Green Master Mix (Yeasen), 0.4 μL of each of the upstream and downstream primers, and 8.7 μL of water. The reaction program is as follows: I: 95 °C for 2 min, II: 95 °C for 10 s, 60 °C for 30 s, and step II is repeated for 40 cycles. The melting curve analysis program is as follows: 95 °C for 15 s, 60 °C for min, and 95 °C for 15 s. The data analysis adopts 2 -ΔΔCT Method. Each sample was repeated three times, and the results are shown in FIG. 3.

[0096] As shown in FIG. 3, the expression levels of the tobacco disease resistance related genes NbHSR203J, NbHIN1, NbWRKY8, NbAcre31, NbWRKY7, and NbPR1 were significantly increased after 12 h of treatment of the tobacco leaves with 5 μM CcHE1 protein.

[0097] Example 4: Plant immune elicitor protein CcHE1 enhances the disease resistance of tobacco

[0098] PBS was inoculated on the back of the leaves as a negative control; 5 μM CcHE1 protein was injected into the tobacco from the back of the leaves, 5 mm of Botrytis cinerea bacterial cake was inoculated on the leaves treated with 5 μM CcHE1 protein after 24 h, and the area of the lesion was counted using image J after 48 h of inoculation. Each sample was repeated three times, and the results are shown in FIG. 4.

[0099] As shown in FIG. 4, compared with the negative control PBS treatment, the lesion area of Botrytis cinerea infection on the tobacco leaves treated with 5 μM CcHE1 was significantly reduced.

[0100] Example 5: Plant immune elicitor protein CcHE1 induces immune response in poplar

[0101] (1) 5 μM CcHE1 protein induces active oxygen burst and callose deposition in poplar

[0102] Active oxygen detection: PBS was used as negative control, and 5 μM CcHE1 treated tobacco was used as positive control. Specifically, poplar leaf discs were immersed in 5 μM CcHE1, vacuumed for 5-10 s, and then placed in a vacuum pump for 15 min. Then, the leaf discs were immersed in 3,3'-diaminobenzidine (DAB) solution (Solarbio) for 12 h, and then decolorized with 95% ethanol for observation.

[0103] Callose deposition detection: After poplar leaves were treated with 5 μM CcHE1 for 15 h, the leaves were completely decolorized and then immersed in aniline blue for 12 h. The deposition of callose was observed and detected using a fluorescence microscope (BX61). Each sample was repeated three times, and the results are shown in FIG. 5.

[0104] As shown in FIG. 5, 5 μM CcHE1 can induce active oxygen burst and callose deposition in poplar leaves.

[0105] (2) 5 μM CcHE1 significantly induced the expression level of poplar resistance-related genes

[0106] After poplar leaves were immersed in 5 μM CcHE1 for 12 h, the samples were collected for detection of the expression level of poplar resistance-related genes. RNA extraction was performed using a RNA extraction kit from TIANGEN according to the instructions, and the RNA content and quality were detected using a Nanodrop.

[0107] Reverse transcription to generate first strand: 2 μg of RNA was used as a template, and cDNA synthesis was performed according to the instructions for using Hifair reverse transcriptase from YEASEN. The reverse transcription product was diluted 4-fold for real-time quantitative PCR reaction to detect the expression level of induced poplar resistance-related genes PtPR1 (sequence as shown in SEQ ID NO. 33) and PtPR4 (sequence as shown in SEQ ID NO. 34).

[0108] The primers used in the real-time fluorescent quantitative PCR reaction are as follows:

[0109] PtActin upstream primer PtActin-5F:

[0110] 5'-TCATCGGAATGGAAGCTGCTGGTA-3' (SEQ ID NO. 21);

[0111] PtActin downstream primer PtActin-3F:

[0112] 5'-TAGTGGAACCACCACTGAGCACAA-3' (SEQ ID NO. 22);

[0113] PtPR1 upstream primer PtPR1-5F:

[0114] 5'-TGGGTTGATGAGAAACCAAAGTATG-3' (SEQ ID NO. 23);

[0115] PtPR1 downstream primer PtPR1-3F:

[0116] 5'-GCTGCACCTTGCTTTAGCAC-3' (SEQ ID NO. 24);

[0117] PtPR4 upstream primer PtPR4-5F:

[0118] 5'-GACGGAGATGGATACGCAAAAGG-3' (SEQ ID NO. 25);

[0119] PtPR4 downstream primer PtPR4-3F:

[0120] 5'-TCGACGATAGAGAGCAGAGGTTTAG-3' (SEQ ID NO. 26).

[0121] The PCR reaction system comprises 0.5 μL of cDNA, 10 μL of qPCR SYBR Green Master Mix (Yeasen), 0.4 μL of each of the upstream and downstream primers, and 8.7 μL of water. The reaction program is as follows: I: 95 °C for 2 min, II: 95 °C for 10 s, 60 °C for 30 s, and step II is repeated for 40 cycles. The melting curve analysis program is as follows: 95 °C for 15 s, 60 °C for min, and 95 °C for 15 s. The data analysis adopts 2 -ΔΔCT Each sample is repeated three times, and the results are shown in FIG. 6.

[0122] As shown in FIG. 6, the expression levels of the disease resistance related genes PtPR1 and PtPR4 of poplar are significantly increased after the poplar leaves are treated with 5 μM CcHE1 protein for 12 h.

[0123] Example 6: Plant immune elicitor protein CcHE1 enhances the disease resistance of poplar leaves to Colletotrichum gloeosporioides

[0124] PBS is used as a control, and 5 μM CcHE1 protein is sprayed or smeared on the poplar leaves. After the leaves are treated with the protein for 24 h, 5 mm Colletotrichum gloeosporioides bacterial cakes are inoculated on the leaves, and the lesion area is counted using image J after 5 d of inoculation. Each sample is repeated three times, and the results are shown in FIG. 7.

[0125] As shown in FIG. 7, compared with the negative control PBS treatment, the lesion area of Colletotrichum gloeosporioides infection on the poplar leaves treated with 5 μM CcHE1 is significantly reduced.

[0126] Example 7: Plant immunity elicitor protein CcHE1 enhances the disease resistance of poplar branches to poplar wood-rot fungus

[0127] The poplar branches were sterilized with 75% alcohol and then scalded, and the wounds were fixed with sealing film after 24 h, serving as the negative control (CK); the poplar branches were sterilized with 75% alcohol and then scalded, and 5 mm fungus cakes of poplar wood-rot fungus were inoculated on the wounds after 24 h, and the fungus cakes were fixed on the wounds with sealing film, serving as the positive control (WT); the poplar branches were sterilized with 75% alcohol and then scalded, 50 μL of 5 μM CcHE1 protein was dropped on the wounds, and 5 mm fungus cakes of poplar wood-rot fungus were inoculated on the wounds after 24 h of absorption, and the fungus cakes were fixed on the wounds with sealing film, serving as the treatment group (5 μM CcHE1). The branches were placed in a ventilated and humid environment for 7 d, and the results were observed, and the lesion area was counted by image J. 15 branches were selected for each treatment, and the results are shown in FIG. 8.

[0128] As shown in FIG. 8, compared with the positive control WT treatment, the lesion area of poplar wood-rot fungus infection on the poplar branches treated with 5 μM CcHE1 was significantly reduced.

[0129] Example 8: Plant immunity elicitor protein CcHE1 enhances the disease resistance of poplar branches to grapevine Botryosphaeria

[0130] The poplar branches were sterilized with 75% alcohol and then scalded, and the wounds were fixed with sealing film after 24 h, serving as the negative control (CK); the poplar branches were sterilized with 75% alcohol and then scalded, and 5 mm fungus cakes of grapevine Botryosphaeria were inoculated on the wounds after 24 h, and the fungus cakes were fixed on the wounds with sealing film, serving as the positive control (WT); the poplar branches were sterilized with 75% alcohol and then scalded, 50 μL of 5 μM CcHE1 protein was dropped on the wounds, and 5 mm fungus cakes of grapevine Botryosphaeria were inoculated on the wounds after 24 h of absorption, and the fungus cakes were fixed on the wounds with sealing film, serving as the treatment group (5 μM CcHE1). The branches were placed in a ventilated and humid environment for 10 d, and the results were observed, and the lesion area was counted by image J. 15 branches were selected for each treatment, and the results are shown in FIG. 9.

[0131] As shown in FIG. 9, compared with the positive control WT treatment, the lesion area of grapevine Botryosphaeria infection on the poplar branches treated with 5 μM CcHE1 was significantly reduced.

[0132] Example 9: Plant immunity elicitor protein CcHE1 enhances the disease resistance of chestnut branches to chestnut blight fungus

[0133] The chestnut branches were sterilized with 75% alcohol and then scalded, and the wounds were fixed with sealing film after 24 h, as negative control (CK); the poplar branches were sterilized with 75% alcohol and then scalded, and 5 mm of chestnut blight fungus cake was inoculated on the wounds and fixed with sealing film after 24 h, as positive control (WT); the poplar branches were sterilized with 75% alcohol and then scalded, 50 μL of 5 μM CcHE1 protein was dropped on the wounds, and 5 mm of chestnut blight fungus cake was inoculated on the wounds and fixed with sealing film after 24 h of absorption, as treatment group (5 μM CcHE1). The above branches were placed in a ventilated and humid environment for 12 d, and the results were observed, and the lesion area was counted by image J. Seven branches were selected for each treatment, and the results are shown in FIG. 10.

[0134] As can be seen from FIG. 10, compared with the positive control WT treatment, the lesion area of chestnut blight fungus infection on the chestnut branches treated with 5 μM CcHE1 was significantly reduced.

[0135] Example 10: Plant immune elicitor protein CcHE1 slows the expansion of grapevine pathogenic fungus Botryosphaeria dothidea on poplar branches

[0136] The poplar branches were sterilized with 75% alcohol and then scalded, and the wounds were fixed with sealing film after 24 h, as negative control (CK); the poplar branches were sterilized with 75% alcohol and then scalded, and 5 mm of grapevine pathogenic fungus cake was inoculated on the wounds, and 50 μL of PBS was dropped on the lesions and fixed with sealing film after 36 h, as positive control (WT); the poplar branches were sterilized with 75% alcohol and then scalded, and 5 mm of grapevine pathogenic fungus cake was inoculated on the wounds, and 50 μL of 5 μM CcHE1 protein was dropped on the lesions and fixed with sealing film after 36 h, as treatment group (5 μM CcHE1). The above branches were placed in a ventilated and humid environment for 8 d, and the results were observed, and the lesion area was counted by image J. Fifteen branches were selected for each treatment, and the results are shown in FIG. 11.

[0137] As can be seen from FIG. 11, compared with the positive control WT treatment, the lesion area of grapevine pathogenic fungus infection on the poplar branches treated with 5 μM CcHE1 was significantly reduced.

[0138] Example 11: Plant immune elicitor protein CcHE1 has no inhibitory effect on the growth of Botrytis cinerea, Ganoderma applanatum, Colletotrichum gloeosporioides, Botryosphaeria dothidea and chestnut blight fungus

[0139] Inoculate the activated Botrytis cinerea, Magnaporthe salvinii, Glomerella cingulata, Botryosphaeria dothidea and Cryphonectria parasitica on the PDA flat plate, when it expands to 1 / 3, punch two holes with 5mm puncher at the two sides of the midpoint of the culture dish, respectively add 50ul of 5uM CcHE1 protein (as the treatment group) and equal amount of PBS (as the control group) into the two holes, and place in the 25℃ incubator for culture, when the fungi grow to the periphery of the holes, take photos for record. Each sample is repeated three times, and the results are shown in Figure 12.

[0140] In the present antibacterial experiment, it can be seen from Figure 12 that 5uM CcHE1 has no antibacterial circle, therefore CcHE1 has no inhibitory effect on Botrytis cinerea, Magnaporthe salvinii, Glomerella cingulata, Botryosphaeria dothidea and Cryphonectria parasitica. That is, CcHE1 does not directly act on the mycelial growth of the above-mentioned pathogenic fungi; according to Examples 3-5, it can be seen that the plant immune elicitor protein CcHE1 achieves the effect of resisting pathogenic fungi by improving the plant immune response.

[0141] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A plant immunity elicitor protein CcHEl, characterized in that, The amino acid sequence of which is shown as SEQ ID NO.

2.

2. The gene encoding the plant immunity elicitor protein CcHEl of claim 1, characterized by, The nucleotide sequence of which is shown as SEQ ID NO.

1.

3. A recombinant vector, expression cassette or recombinant bacteria containing the encoding gene of claim 2.

4. Use of the plant immune elicitor CcHE1 of claim 1 or the encoding gene of claim 2 or the recombinant vector, expression cassette or recombinant bacteria of claim 3 in inducing plant defense response or improving plant disease resistance.

5. Use according to claim 4, characterized in that, The plant is poplar, tobacco or chestnut.

6. Use according to claim 4 or 5, characterized in that, The disease resistance is against diseases caused by Botrytis cinerea, Ganoderma boninense, Glomerella cingulata, Elsinoe ampelina and Cryphonectria parasitica.

7. A method of controlling plant diseases, characterized by, The plant is treated with the plant immune elicitor CcHE1 of claim 1.

8. The method of claim 7, wherein, The specific treatment is that the plant immune elicitor CcHE1 is sprayed or smeared on the surface of the plant or injected into the leaves of the plant.

9. The method according to claim 7 or 8, characterized in that, The plant is poplar, tobacco or chestnut.

10. The method of claim 7, wherein, The plant disease is caused by Botrytis cinerea, Ganoderma boninense, Glomerella cingulata, Elsinoe ampelina and Cryphonectria parasitica.

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