CN110590917A - Pseudomonas aeruginosa flagellin for improving plant disease resistance and coding gene and application thereof - Google Patents

Abstract

The invention discloses pseudomonas aeruginosa flagellin for improving plant disease resistance, and a coding gene and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO:1 or SEQ ID NO:1, one or more amino acids are substituted, deleted and/or added, and the amino acid sequences of the proteins with the same function are expressed; the nucleotide sequence of the gene for coding the protein is shown as SEQ ID NO:2 or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein. The protein can be used as an immune exciton, can obviously improve the resistance disease of plants, has low use concentration and quick response, and can effectively reduce the agricultural production cost.

Description

Pseudomonas aeruginosa flagellin for improving plant disease resistance and coding gene and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to pseudomonas aeruginosa flagellin for improving plant disease resistance, and a coding gene and application thereof.

Background

At present, a plurality of biopesticide products are widely applied in the world, and the most extensive biopesticide products are agricultural antibiotics such as bacillus thuringiensis (Bt) and validamycin, zhongshengmycin, brassin and the like. The products achieve the aims of insect resistance and disease resistance through bacteriostatic and insecticidal substances of biological origin. Compared with chemical pesticide and antibiotic agent, these products have short degradation period and high environment compatibility. However, the occurrence of diseases and insect pests is the process of interaction between plants and pathogens, and in the past, the research and development of biopesticides often neglects the role of the immunity of plants in the process of resisting the diseases and insect pests of the plants. In recent years, with the research progress of plant disease resistance mechanism, the plant immunity induction technology becomes a new bright point in the development of biological pesticides. The research on microbial protein pesticides with the functions of stimulating plant immunity and disease resistance, promoting growth and increasing yield has attracted extensive attention and attention at home and abroad. As early as 2001, the american company EDEN developed the Messenger pesticide product from allergenic proteins of bacterial origin, however its nature is still a fungicide. In 2016, the subject group of professor Qiudens of plant protection institute of agricultural academy of China extracted an activator protein from alternaria tenuissima, and the protein is proved to greatly improve the resistance of plants to virus diseases, so that the first global plant immunity protein pesticide, namely the atrazine, is developed, the disease resistance and yield increase of agricultural products are realized by enhancing the autoimmunity of the plants, and the pioneer of 'plant immunity' is created.

However, from the macroscopic view, the plant immunity pesticide on the market has fewer varieties, and only has a plurality of varieties such as Tailing and Harpin protein. At a microscopic level, these products still have limitations. Firstly, receptors of alternaria tenuissima activator protein and Harpin protein are not clear, which means that the alternaria tenuissima activator protein and Harpin protein are difficult to use, scientific and effective guidance is made in theory, the application range and the use concentration of the alternaria tenuissima activator protein and the Harpin protein can be judged only through experimental experience, and a long-term continuous exploration process is needed; secondly, the sensitivity of the plants to the plant immunity pesticides is low, the final using concentration is 100 mu mol, the using amount per mu is 100g, and the cost is over 40 yuan per mu. In order to reduce the cost of agricultural production, research and development of new products is still required.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides pseudomonas aeruginosa flagellin for improving plant disease resistance, a coding gene and application thereof.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

the pseudomonas aeruginosa flagellin for improving the disease resistance of the plants has an amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:1 is substituted, deleted and/or added with one or more amino acids, and expresses the amino acid sequence of the same functional protein.

The nucleotide sequence of the gene for coding the pseudomonas aeruginosa flagellin is shown as SEQ ID NO. 2, or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein.

A recombinant vector comprising the gene encoding pseudomonas aeruginosa flagellin as described above.

A recombinant bacterium comprising the gene encoding Pseudomonas aeruginosa flagellin as described above.

A host cell comprising the gene encoding Pseudomonas aeruginosa flagellin as described above.

The application of the pseudomonas aeruginosa flagellin in improving the disease resistance of plants and inducing the defense reaction of the plants comprises the following specific steps: the application of the composition in improving the resistance of plants to pseudomonas putida infection, rice blast infection and fusarium graminearum infection is further disclosed, and the plants are cruciferous plants (such as arabidopsis thaliana, rape and the like), solanaceous plants (such as tobacco, tomato and the like) and gramineous plants (such as rice, corn, wheat and the like). More specifically: the pseudomonas aeruginosa flagellin is applied to the improvement of the infection resistance of cruciferae and solanaceae plants to pseudomonas syringae, the improvement of the infection resistance of rice blast germs and the improvement of the infection resistance of corn and wheat to fusarium graminearum.

When the Pseudomonas aeruginosa flagellin is used in the above application, the concentration is 0.1-2. mu.M, preferably 1. mu.M.

In addition, pseudomonas aeruginosa flagellin can be prepared into a plant disease resistance medicament for preventing and treating diseases in agricultural production, so that the use cost in agricultural production is effectively reduced.

The pseudomonas aeruginosa flagellin for improving the disease resistance of plants, the coding gene and the application thereof provided by the invention have the following beneficial effects:

the pseudomonas aeruginosa flagellin serving as an immune exciton can obviously improve the resistance disease of plants, has low use concentration and quick response, and can effectively reduce the agricultural production cost.

Drawings

FIG. 1 shows the experimental results of the optimization of protein expression conditions.

FIG. 2 shows the results of the solubility analysis and purification of the protein.

FIG. 3 shows the callose detection results of the protein-stimulated plant immunity.

FIG. 4 shows the detection results of protein-stimulated disease-resistant genes.

FIG. 5 shows the experimental results of protein inhibition of Pseudomonas syringae infection in Arabidopsis thaliana.

FIG. 6

a is the actual measurement comparison result of the growth of corn infected by fusarium graminearum with protein inhibition

b is the experimental result of inhibiting corn infection by fusarium graminearum with protein

FIG. 7

a is the actual measurement comparison result of the growth of rice infected by rice blast germs inhibited by protein

b is the experimental result of inhibiting rice blast germs from infecting rice by protein

Detailed Description

The experimental materials used in the following examples are as follows:

1. rice material

Rice (OryzasativaL.): short grain japonica rice variety Nipponlily (NPB) (NPB is an international universal variety that has been sequenced from the whole genome).

2. Corn material:

the dredge sheet 20 is purchased by a seed company.

3. Arabidopsis thaliana material

Columbia wild type Arabidopsis thaliana col-0 was preserved by the subject group of Chua professor, institute of Life sciences, Sichuan university of agriculture.

4. Strain material:

pseudomonas syringae DC3000 is preserved by the subject group of Chua Yi professor of the institute of Life sciences of Sichuan university, rice blast ZB15 race material is given to the rice research institute of Sichuan university, and corn fusarium graminearum material is given to the college of Sichuan university.

Example 1 construction of Flagellin expression vectors

A gene sequence of a flagellin gene of pseudomonas aeruginosa is analyzed by using bioinformatics software Geneius R9, restriction enzyme cutting sites of a pET-28b prokaryotic expression vector are combined, and an upstream primer with NdeI cutting sites and a downstream primer with XholI cutting sites are designed according to the principle of open reading frame ORF.

A forward primer: 5' -GACCATATGATGGCTCTTACTGTTAAT-3' (SEQ ID NO:3), wherein the digestion sequence of NdeI is underlined;

reverse primer: 5' -TCACTCGAGTTATCTAAGCAAAGACAACACAGA-3' (SEQ ID NO:4), in which the XholI cleavage sequence is underlinedAnd (4) columns.

Amplifying the full-length flagellin gene by PCR, wherein the amplification program comprises the following steps: 30s at 98 ℃; 34 cycles of 98 ℃ for 10s, 55 ℃ for 10s, 72 ℃ for 30 s; 2min at 72 ℃; storing at 4 ℃.

And recovering the PCR product, connecting the PCR product to a vector, and screening and identifying to obtain the recombinant plasmid.

And carrying out double enzyme digestion on the recombinant plasmid and a prokaryotic expression vector pET-28b (+), recovering and purifying, connecting the recombinant plasmid and the prokaryotic expression vector by using ligase overnight, transforming the recombinant plasmid into escherichia coli BL21(DE3) by adopting a heat shock method, selecting positive clone, shaking the bacterium, extracting the plasmid, carrying out enzyme digestion and sequencing verification, and obtaining the recombinant plasmid pET-28b-flagellin expression vector.

Example 2 protein expression Condition optimization

The recombinant plasmid pET-28b-flagellin expression vector is transformed into Escherichia coli BL21(DE3) by a heat shock method to obtain recombinant Escherichia coli containing the recombinant plasmid pET-28b-flagellin, and the recombinant Escherichia coli is named as BL21(DE3)/pET-28 b-flagellin. The plasmid extraction and transformation of the expression host strain were performed as in example 1, and expression was performed after verification. The correct expression strains were verified for overnight activation, while the strains containing empty pET-28b plasmid were used as controls. Adding 1mL of overnight culture solution into 100mL LB liquid medium (1% inoculum size) containing 100. mu.g/mL kanamycin, shaking-culturing at 37 deg.C and 200r/min for 2-3h to OD₆₀₀Is 0.6-0.8. At this time, a gradient of the concentration of the inducer IPTG (final concentrations of 0.1mmol/L, 0.3mmol/L, and 0.5mmol/L of IPTG were added), and a temperature gradient (induced expression temperatures of 25 ℃ C., 28 ℃ C., and 37 ℃ C., respectively) were set, and an orthogonal experiment was conducted to search for the optimum expression conditions. And continuously carrying out shaking culture for 12h at 220r/min under the conditions, inducing and expressing the target protein, then carrying out high-speed centrifugation, collecting thalli and adding a buffer solution. After the thalli is broken by ultrasonic, the thalli is centrifuged at high speed at 4 ℃, and the supernatant is collected to obtain the expression protein liquid. 20 mu L of supernatant was taken, 5. mu.L of 5 XSDS loading buffer (denaturation) was added to resuspend the cells, heated in boiling water bath for 10min, centrifuged at 13000R/min for 10min, the supernatant was taken for SDS-PAGE detection, stained with Coomassie blue R250, and the expression was observed as shown in FIG. 1. The label in figure 1 is temperature + IPTG concentration (mM).

As can be seen from FIG. 1, when the bacterial solution was cultured to OD₆₀₀The value is 0.6, the final concentration of the IPTG is 0.5mmol/L, and the protein expression is the best condition when the induction culture is carried out for 12 hours under the condition of 28 ℃.

EXAMPLE 3 expression and purification of proteins

1. BL21(DE3)/pET-28b-flagellin was inoculated in LB liquid medium and cultured overnight at 37 ℃ with shaking at 200 rpm/min.

2. Protein expression was induced as shown in example 2, using the conditions: transferring the overnight culture solution to LB liquid medium containing 100. mu.g/mL kanamycin at a volume ratio of 1:100, and culturing at 37 ℃ and 200rpm/min with shaking to OD₆₀₀The concentration was 0.6, and then 0.1mmol/LIPTG was added thereto, and the mixture was shake-cultured at 30 ℃ and 200rpm/min for 12 hours to collect the cells.

3. And (3) taking the thalli obtained in the step (2), carrying out ultrasonic disruption after resuspension by using a PBS (phosphate buffer solution) (the parameters of ultrasonic disruption: total ultrasonic time is 30min, stopping for 5s every 5s, and power is 200W), then carrying out centrifugation at 12000rpm for 10min, and respectively collecting supernatant and sediment.

4. The supernatant obtained in step 3, the precipitate obtained in step 4, and the cells not induced by IPTG were subjected to SDS-PAGE, and the results are shown in FIG. 2.

In fig. 2: lane 1 is Marker; lane 2 is the supernatant after disruption and centrifugation; lane 3 is pellet after disruption and centrifugation; lane 4 is the impurity wash during protein purification. Lane 5 is the final purified flagellin protein sample. As can be seen from FIG. 2, following IPTG induction of BL21(DE3)/pET-28b-flagellin, the expressed flagellin protein was present as both soluble protein (44kDa) and inclusion body protein.

5. Taking the supernatant obtained in the step 4, and adopting a His tag protein purification kit (soluble protein) (Kangjie company product, product code CW0894), specifically operating as follows:

filling 5ml of Ni-Agarose filler into an affinity column, slowly flowing the supernatant obtained in the step 4 through the affinity column, eluting 6 column volumes by using PBS buffer solution containing 10mM imidazole to remove impurities, eluting 5 column volumes by using PBS buffer solution containing 500mM imidazole, and collecting the eluates after passing through the column, thereby obtaining the flagellin protein solution. The protein concentration in the flagellin protein solution was 1.2mg/mL, as determined by BCA protein concentration assay kit (Solarbio, catalog number PC0020-500 microwell (50T)).

6. 20 mu.L of each supernatant of the supernatant and the precipitate resuspension and the purified protein are respectively added with 5 mu.L of 5 xSDS loading buffer (denaturation) to resuspend the thalli, the thalli are heated in boiling water bath for 10min, centrifuged at 13000R/min for 10min, and the supernatant is taken for SDS-PAGE detection and stained by Coomassie Brilliant blue R250, and the result is shown in figure 2.

As can be seen from FIG. 2, an expressed histidine-containing protein (His-flagllin) having a molecular weight of about 42-kDa was obtained after purification by SDS-PAGE.

Example 4 detection experiment of the stimulation of plant Immunity by the protein Flagellin

The arabidopsis thaliana and the rape are cruciferae plants, and the flagellin causes resistance reaction of the arabidopsis thaliana and has guiding significance for the use of the flagellin on the rape. The specific experimental process of arabidopsis thaliana as an experimental object is as follows:

(1) expression of the protein flagellin causes a Reactive Oxygen Species (ROS) immune response in plants

Reactive Oxygen Species (ROS) are important means for early plant immunity, i.e., the production of some substances with oxidative activity, such as H, at the site of infection₂O₂Oxygen anions and the like, and achieve the aims of killing virus and inhibiting bacteria through the oxidation effect of the oxygen anions. Adjusting the final concentration of the flagellin protein to be 1 mu M, taking water as a blank control, infecting arabidopsis leaves of four weeks old by a syringe permeation method, and treating for 4 h. Collecting the treated leaves, placing into a six-hole plate, adding 2ml of 0.5mg/ml DAB staining solution, dyeing for six hours, replacing eluent (mixture of alcohol and acetic acid with volume ratio of 3: 1), decolorizing twice in 95 deg.C water bath for 15min each time, replacing eluent, standing for 30min, and directly observing. Three biological replicates per treatment were used and the results are shown in figure 3.

(2) Expression of the protein flagellin to cause a callose-accumulating immune response in plants

The cells produce callose accumulation to strengthen the mechanical strength of cell walls and block the channels for pathogen diffusion between cells, thereby limiting further pathogen invasion. Adjusting the final concentration of the flagellin protein to be 1 mu M, taking water as a blank control, infecting arabidopsis thaliana leaves of four weeks old by an injector permeation method, treating for 12h, collecting the treated leaves, putting the treated leaves into a six-hole plate, adding a proper amount of eluent, and horizontally incubating for 4 h. And replacing the eluent with aniline blue staining solution with the final concentration of 0.1mg/ml, keeping out of the light, dyeing at room temperature for 1h, and observing the callose accumulation condition through a fluorescence microscope. Three biological replicates per treatment were used and the results are shown in figure 3.

(3) Expression of the protein flagellin causes a Programmed Cell Death (PCD) immune response in plants

Sample treatment was as above, but only the left part of the veins of arabidopsis thaliana leaves were stained, and the veins were measured right as a blank. After 24 hours of treatment, trypan blue staining solution with the final concentration of 0.1mg/ml is added to boil for 2min and the mixture is kept stand and stained for 8 hours at room temperature. After 3 days of decolorization with 1g/ml of trichloroethanol, cell death was directly observed. Three biological replicates per treatment were used and the results are shown in figure 3.

In FIG. 3, A is the protein-stimulated active oxygen burst immune response in plants; b is protein-stimulated apoptosis immune response of the plant; c is the callose accumulation immune response of protein-stimulated plants.

As shown in FIG. 3, the expression protein flagellin can cause plant Reactive Oxygen Species (ROS) immune reaction, callose accumulation immune reaction and cell programmed death (PCD) immune reaction, namely, resistance reaction of plants, and further can be used for preventing and treating agricultural production diseases.

Example 5 expression of the protein flagellin causes expression of Arabidopsis-related disease resistance genes

Preparing an arabidopsis protoplast by an enzymatic hydrolysis method, and transforming a disease-resistant related reporter gene FRK1 into the arabidopsis protoplast by a PEG (polyethylene glycol) transformation method. Adding 1 mu M of expressed protein flagellin and water into an arabidopsis thaliana protoplast culture solution, culturing at 25 ℃ and 2200Lux for 12h, centrifuging, collecting the cultured protoplast, treating by using a glucuronidase and luciferase reporter gene detection kit, and quantitatively detecting the enzyme activity of the glucosidase and luciferase by using an enzyme labeling instrument, wherein each treatment is three biological repetitions, and the specific operation process is as follows:

adding 50 mul of protoplast cell lysate into an EP tube, and placing the tube on a vortex oscillator to oscillate and lyse the protoplast cells.

And adding 5 mul of the protoplast cell lysate obtained in the step I and 100 mul of Luc working solution into a 96-well plate in sequence, and quantitatively detecting the activity of the luciferase by using a microplate reader.

Adding 2 μ L of the protoplast cell lysate obtained in the step I and 250 μ L of MuG working solution into a 96-well plate in sequence, standing at 37 ℃ for 0.5h, adding 225 μ L of 0.2mol/L Na₂CO₃The reaction was terminated and the activity of glucuronidase was quantitatively measured with a microplate reader.

The Luc value is expressed as LAV (LAV); gus values are expressed as GAV (GAV); the relative expression intensity of FRK1 gene was calculated by the following formula:

relative expression intensity of FRK1 Gene ═ LAV/GAV (1000X)

The result of the expression protein flagellin stimulating the expression of the arabidopsis related disease-resistant gene is shown in fig. 4.

As can be seen from fig. 4, the relative expression intensity of FRK1 gene in water-treated arabidopsis thaliana protoplast was 0.22, the relative expression intensity of FRK1 gene in flagellin-treated arabidopsis thaliana protoplast was 1.18, the relative expression level of FRK1 gene related to disease resistance was up-regulated by 5.33 times, and flagellin significantly activated the plant immune response.

Example 6 Arabidopsis thaliana in vivo anti-DC 3000 experiment

Inoculating Pseudomonas syringae DC3000 thallus into 20ml SOC + str (streptomycin) liquid culture medium, culturing at 28 deg.C overnight for 14-16h, and determining its OD₆₀₀Values, gradient diluted to OD 0.00005. Four-week-old healthy Arabidopsis leaves were injected, one 4 leaves were sampled on day 0 and designated as T0, and two leaves were sampled on day 3 and designated as T3. Each leaf was punched with a punch, and a small punched round piece was taken and added 500. mu.l of 10mM MgCl for T0 days₂Grinding in 1.5ml ep tube, treating four samples in the same way, taking 50. mu.l of each spot on the same SOC + str (streptomycin) solid plate (the plate must be dried to keep the spot shape), 2Culturing at 8 ℃ for 16-24h, photographing and observing, and counting the growth condition of colonies. Two leaves of 5 plants each at T3 day were sampled, and small round pieces were punched out and 250. mu.l of 10mM MgCl was added₂Ground in 1.5ml ep tube and mixed with MgCl₂Diluting to 1 × 10^-5. Five samples of the same treatment, including diluted samples (one for 30 samples), were spotted at 10. mu.l each on the same SOC + str (streptomycin) solid plate (using a square dish, the plate must be dried to keep the spotted shape), incubated at 28 ℃ for 16-24h, photographed for observation, and the colony growth was counted, the results are shown in FIG. 5.

As seen from FIG. 5, the amount of growth of Pseudomonas syringae DC3000 was suppressed by 29.8% in 1. mu.M flagellin-treated Arabidopsis thaliana compared to that in the untreated Arabidopsis thaliana.

In addition, pathogenic bacteria causing diseases of arabidopsis thaliana, tobacco and tomato are all DC3000, and the arabidopsis thaliana added with flagellin can inhibit the growth of DC3000, which can be used as reference for diseases of tobacco and tomato caused by DC 3000.

Example 7 expression of the protein flagellin induces plants to improve disease resistance

(1) Increasing resistance of corn to sickle of cereal grain

Inoculating Fusarium graminearum mycelium into CMC liquid culture medium, culturing at 25 deg.C in dark for 3-7 days, filtering with gauze, centrifuging at 10000rpm/10min to collect spores, counting the number of spores with hemocytometer, and adjusting concentration to 2 × 10⁵And storing in a refrigerator at 4 deg.C (for one month).

Two-week-old corn leaves are taken, the middle part is cut into 5cm in length and soaked in 1 mu g/ml auxin 6BA solution, and 12-13 leaves are taken for each treatment. Flagellin protein was added at a final concentration of 1. mu.M, with auxin 6BA solution as a blank. Respectively and uniformly dripping fusarium graminearum spore liquid on leaves, culturing for 3-4 days under the conditions of 28 ℃, 12h of illumination and 12h of darkness, and observing the morbidity. The area percentage of lesions was counted by imageJ software and the results are shown in fig. 6 and 7.

As can be seen from FIGS. 6a and 6b, the protein elicitor significantly improves the resistance of wheat to Fusarium graminearum, and the infection rate of Fusarium graminearum is reduced by 58%.

(2) Increasing resistance of rice to rice blast

Inoculating Pyricularia oryzae on CM solid culture medium, culturing at 28 deg.C for 13-15 days, scraping off mycelia with gun head, washing with 5-10ml of sterile water, filtering with gauze to 50ml centrifuge tube, centrifuging at 10000rpm for 10min, collecting spores, counting number of spores with blood counting plate, adjusting concentration to 1 × 10⁶Storing at normal temperature (within one week).

Taking four-week-old rice leaves, cutting the middle part into 5cm length, soaking in 1 μ g/ml auxin 6BA solution, and taking 12-13 leaves for each treatment. Flagellin protein was added at a final concentration of 1. mu.M, with auxin 6BA solution as a blank. Respectively and uniformly dripping the rice blast fungus spore liquid on leaves, culturing for 3-4 days under the dark condition of 12h illumination at 28 ℃, and observing the disease occurrence condition. The area percentage of lesions was counted by imageJ software and the results are shown in fig. 8 and 9.

As can be seen from FIGS. 7a and 7b, the protein elicitor significantly improves the resistance of rice to rice blast, and reduces the infection rate of rice blast germs by 71%.

Sequence listing

<110> Chengdu Luxinuo Biotechnology Co., Ltd

Sichuan university of agriculture

<120> pseudomonas aeruginosa flagellin for improving plant disease resistance, and coding gene and application thereof

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Ser Thr Gly Ser Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Leu

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Gln Ile Ala Asn Arg Leu Thr Ser Gln Val Asn Gly Leu Asn Val Ala

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Thr Lys Asn Ala Asn Asp Gly Ile Ser Leu Ala Gln Thr Ala Glu Gly

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Claims (10)

1. The pseudomonas aeruginosa flagellin for improving the disease resistance of plants is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO:1, or SEQ ID NO:1 is substituted, deleted and/or added with one or more amino acids, and expresses the amino acid sequence of the same functional protein.

2. The gene encoding pseudomonas aeruginosa flagellin for improving plant disease resistance as claimed in claim 1, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 2, or SEQ ID NO:2 is substituted, deleted and/or added with one or more nucleotides, and can code the nucleotide sequence of the same functional protein.

3. A recombinant vector comprising the gene of claim 2.

4. A recombinant bacterium comprising the gene according to claim 2.

5. A host cell comprising the gene of claim 2.

6. Use of pseudomonas aeruginosa flagellin as claimed in claim 1 for enhancing disease resistance and inducing plant defense response.

7. The application of claim 6, in particular to the application of improving the resistance of plants to pseudomonas syringae infection, rice blast fungus infection and fusarium graminearum infection.

8. Use according to claim 6 or 7, wherein the plant is a crucifer, a solanaceous plant and a graminaceous plant.

9. The use according to claim 6, wherein the Pseudomonas aeruginosa flagellin is used at a concentration of 0.1-2 μ M.

10. A disease resistance drug for plants comprising the pseudomonas aeruginosa flagellin of claim 1.