Use of a pacap polypeptide as a plant immunomodulator

Abstract

This invention discloses the application of PACAP peptides as plant immunomodulators, belonging to the field of plant immunology technology. This invention is the first to apply PACAP to a plant system, discovering that it can act as a novel plant immunomodulator to regulate plant immune responses, thereby enhancing plant resistance to pathogenic microorganisms. Experimental results show that PACAP can rapidly induce intracellular calcium ion influx in Arabidopsis thaliana cells, activate the MAPK signaling pathway, and upregulate the expression of early immune marker genes such as FRK1. Furthermore, PACAP treatment significantly improves plant resistance to pathogens such as *Pseudomonas syringae* and *Ralstonia solanacearum*. This invention also confirms that not only does the full-length PACAP (1-38) possess plant immune-promoting activity, but its N-terminal truncated peptide (1-27) and receptor antagonist fragment (6-38) also have plant immune-inducing functions. This invention provides a new technical solution for the development of plant immune inducers.

Description

Technical Field

[0001] This invention belongs to the field of plant immune technology, specifically relating to the application of PACAP peptides as plant immune modulators. Background Technology

[0002] For a long time, chemical control has been the primary means of controlling crop diseases in agricultural production. This involves spraying chemical pesticides to directly target and rapidly kill pathogens, thereby controlling disease occurrence. However, due to the large dosages and improper application methods of pesticides, problems such as environmental pollution, crop damage, and increased pesticide resistance in pathogens are easily caused. This not only disrupts the balance of the agricultural ecosystem but also poses potential threats to food safety and human health. Therefore, developing safe, efficient, and environmentally friendly new plant disease control technologies has become an urgent need for sustainable agricultural development.

[0003] Studies have shown that, similar to animals, plants possess a sophisticated immune defense system capable of sensing pathogenic microorganisms and activating their own defense responses. Plant immunity is not achieved by directly killing pathogens, but rather by regulating the plant's internal defense and metabolic networks, inducing the expression of disease-resistance-related genes, thereby enhancing the plant's overall resistance to pathogens.

[0004] Over a long period of evolution, plants have developed two interconnected immune systems: pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is activated by pattern recognition receptors (PRRs) located on the cell membrane surface, which recognize pathogen-associated molecular patterns (PAMPs). These PAMPs include, but are not limited to, bacterial flagellin, elongation factors, fungal chitin polysaccharides, xylanases, and endogenous stimulating peptides. PTI activation triggers a series of immune responses, including calcium ion influx, callose deposition, reactive oxygen species (ROS) generation, stomatal closure, and the production of plant hormone signals such as salicylic acid and jasmonic acid, thus playing a defensive role in the early stages of pathogen infection.

[0005] To overcome the plant's protective barrier against PTIs, some successfully infected pathogens secrete effector factors into plant cells, suppressing the host's immune response. In response, plants have evolved intracellular immune receptors, such as nucleotide-binding leucine-rich repeat receptors (NLRs), to recognize specific effector factors and activate ETIs. Compared to PTIs, ETIs typically induce a stronger and more durable immune response, often accompanied by programmed cell death, resulting in a hypersensitive response (HR) to limit pathogen spread.

[0006] Once activated, plant immune responses are often systemic, broad-spectrum, and persistent, extending from local tissues to the entire plant, thus effectively defending against various diseases. Based on these characteristics, substances capable of activating plant immune responses have been developed as plant immune inducers for the control of plant diseases. Currently reported plant immune inducers mainly include sugars, glycopeptides, lipids, proteins, secondary metabolites, and nucleotides. However, existing immune inducers still have limitations in terms of variety, mechanism of action, and application scope, necessitating the discovery of new immune-stimulating substances.

[0007] Pituitary adenylate cyclase activating polypeptide (PACAP) is a short peptide organic compound first isolated from sheep hypothalamus tissue by Miyata et al. in 1989, and named for its ability to activate adenylate cyclase. PACAP belongs to the vasoactive intestinal peptide (VIP) family and has high sequence homology with VIP. Currently, research on PACAP mainly focuses on the animal and medical fields; its function in plant systems has not been publicly reported. Summary of the Invention

[0008] To address the problems existing in the prior art, one of the technical problems to be solved by the present invention is to provide a natural PACAP polypeptide that can regulate the plant immune system. Another technical problem to be solved by the present invention is to provide the application of the aforementioned PACAP polypeptide in improving plant disease resistance.

[0009] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0010] The application of a PACAP polypeptide in the preparation of a plant immune modulator, wherein the PACAP polypeptide has the activity of enhancing plant immune response.

[0011] In some embodiments, the PACAP polypeptide is selected from: the full-length PACAP1-38 amino acid sequence as shown in SEQ ID NO. 1, the PACAP1-27 fragment as shown in SEQ ID NO. 2, or the PACAP6-38 fragment as shown in SEQ ID NO. 3.

[0012] In some embodiments, the plant immunomodulator is used to activate the plant's immune response.

[0013] In some embodiments, the activation of the plant's immune response manifests as one or more of the following: inducing the expression of immune marker genes FRK1 and WRKY29, promoting phosphorylation of MAPK proteins, or triggering intracellular calcium ion influx.

[0014] In some embodiments, the plant immunomodulator is used to enhance plant resistance to bacterial pathogens.

[0015] In some embodiments, the bacterial pathogens are *Pseudomonas syringae* and *Ralstonia solanacearum*.

[0016] A plant immunomodulator comprising PACAP with an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, and an agronomically acceptable carrier and / or adjuvant.

[0017] In some embodiments, the regulator is a wettable powder, an aqueous solution, or an emulsifiable concentrate.

[0018] A method for enhancing plant immune responses includes the step of applying an effective amount of the plant immune modulator to the plant or its growing environment.

[0019] A method for controlling plant diseases caused by *Pseudomonas syringae* or *Ralstonia solanacearum* is to apply an effective amount of PACAP, with an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, to plants, wherein the plants are *Arabidopsis thaliana* or tobacco.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] This invention is the first to apply PACAP to a plant system, discovering that it can serve as a novel plant immunomodulator to regulate plant immune responses and thereby enhance plant resistance to pathogenic microorganisms. Experimental results show that PACAP can rapidly induce intracellular calcium ion influx in Arabidopsis cells, activate the MAPK signaling pathway, and upregulate the expression of early immune marker genes such as FRK1. Furthermore, PACAP treatment significantly improves plant resistance to *Pseudomonas syringae* and *Ralstonia solanacearum*. The results also indicate that not only does the full-length PACAP (1-38) possess plant immune-promoting activity, but its N-terminal truncated peptide (1-27) and receptor antagonist fragment (6-38) also have plant immune-inducing functions, providing a new technical solution for the development of plant immune inducers.

[0022] The PACAP provided by this invention can activate plant immune responses and has clear immune-inducing activity; moreover, PACAP is a short peptide substance of natural origin, which is environmentally friendly and does not easily cause environmental pollution; PACAP works by activating the plant's own basic immune system, and has the characteristics of broad spectrum and low inducing pathogen resistance, making it suitable for green control of plant diseases.

[0023] This application successfully developed a novel and highly efficient plant immune inducer, which has good application value in agriculture and forestry. Attached Figure Description

[0024] Figure 1 Image showing GUS staining results of pFRK1::GUS transgenic Arabidopsis treated with PACAP;

[0025] Figure 2 The expression diagram of PACAP activating the Arabidopsis immune marker genes FRK1 and WRKY29;

[0026] Figure 3 A PACAP-induced calcium ion influx map in Arabidopsis thaliana;

[0027] Figure 4 A diagram showing the phosphorylation of Arabidopsis MAPKs proteins activated by PACAP;

[0028] Figure 5 Figure showing the results of PACAP in improving the resistance of Arabidopsis thaliana to Pseudomonas syringae.

[0029] Figure 6 Figure showing the results of PACAP in improving tobacco resistance to Ralstonia solanacearum. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] The PACAP 1-38, PACAP 1-27, and PACAP 6-38 (≥95% purity) used in this embodiment were synthesized by GenScript (Nanjing, China) and stored in sterile water. 1-38, 6-38, and 1-27 represent different truncation forms of PACAP. PACAP 1-38 is the full-length short peptide, PACAP 6-38 is a truncation starting from the sixth amino acid at the N-terminus, and PACAP 1-27 is a truncation of 27 amino acids from the head to the N-terminus. The amino acid sequences are shown below:

[0032] PACAP 1-38:HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK(.SEQ ID NO. 1);

[0033] PACAP 1-27:HSDGIFTDSYSRYRKQMAVKKYLAAVL(SEQ ID NO. 2);

[0034] PACAP 6-38: FTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO. 3).

[0035] Example 1: PACAP promotes the expression of immune marker genes

[0036] 1. Construction of pFRK1::GUS transgenic Arabidopsis thaliana

[0037] This invention uses Arabidopsis thaliana as the experimental subject and employs biochemical screening methods. The FRK1 gene is a known marker gene for early immune responses, which can reflect the level of plant immune responses to a certain extent. First, the promoter of the FRK1 gene was cloned using high-fidelity polymerase (Phanta Super-Fidelity DNA Polymerase, Nanjing Novizan Biotechnology Co., Ltd.). The cloning primers were synthesized by Nanjing Qingke Biotechnology Co., Ltd., and their sequences are as follows (underlined primers are Gateway system adapter primers, and the remaining parts are promoter sequence primers). The PCR amplification system and program are shown in Tables 1 and 2 below:

[0038] FRK1-Pro-attbF: GGGG ACA AGT TTG TAC AAA AAA GCA GGC TTC GCTGTCGAACATACATTGTCGC;

[0039] FRK1-Pro-attbR: GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CGATGATCCGCTTTCAACGATAC.

[0040] Table 1. Preparation system for high-fidelity enzyme PCR

[0041]

[0042] Table 2 High-fidelity enzyme PCR running procedure

[0043]

[0044] The promoter sequence was constructed into the pDonr207 entry vector of the Gateway system. The reaction mixture consisted of 1 μL of vector, 1 μL of PCR product, and 0.4 μL of BP enzyme (Thermo Fisher Scientific), and was incubated overnight. The next day, 0.4 μL of proteinase K (Sangon Biotech) was added to the reaction mixture, and the mixture was incubated at 37°C for 5 minutes. The reaction mixture was then added to DH5α *E. coli*, and subjected to a series of incubation cycles: ice bath for 30 minutes, water bath for 90 seconds, and ice bath for 2 minutes. Subsequently, 500 μL of liquid LB medium was added to the tube, and the mixture was shaken at 37°C for one hour. A 50 mg / mL gentamicin sulfate solution (Sangon Biotech) was prepared, filtered, and sterilized to serve as a 1000-fold stock solution of antibiotic (the gentamicin sulfate used below is the same as here). Solid LB medium containing the antibiotic was prepared, and the bacterial culture was evenly spread on plates and incubated in the dark at 37°C for one day. Subsequently, the *E. coli* bacteria grown on the culture medium were picked and transferred into clear 96-well shake plates containing liquid LB. PCR identification was performed using Kangwei Century 2×Es TaqMasterMix (Dye) enzyme. The identification primers are as follows, and the PCR system and procedure are shown in Tables 3 and 4 below:

[0045] attB1: GGGGACAAGTTTGTACAAAAAA(5'-3');

[0046] attB2: GGGGACCACTTTGTACAAGAA(5'-3').

[0047] Table 3 Taq enzyme identification preparation system

[0048]

[0049] Table 4 Taq enzyme identification procedure

[0050]

[0051] For bacterial cultures with correct band sizes, aspirate 10 μL and add 3-4 mL of LB liquid containing gentamicin sulfate. Incubate overnight, then extract plasmids for sequencing the following day (Kangwei Century Biotechnology Co., Ltd.). For plasmids with correct sequencing, proceed to the next step of the LR reaction. The reaction system consists of 1 μL of pGWB535 vector, 1 μL of the correctly sequenced pDonr207 vector, and 0.4 μL of LR enzyme (Thermo Fisher Scientific Co., Ltd.). Subsequent steps are the same as for constructing the pDonr207 vector: bacterial selection and identification, culture, and plasmid extraction for sequencing. After obtaining the pGWB535 vector containing the FRK1 promoter sequence, it was transformed into GV3101 Agrobacterium tumefaciens cells. The transformation procedure was as follows: 2 μL of pGWB535 vector was added to GV3101 competent cells, followed by sequential incubation on ice for 5 minutes, freezing in liquid nitrogen for 5 minutes, water bath at 37°C for 5 minutes, and then on ice for 5 minutes. Finally, 500 μL of liquid LB was added, and the cells were shaken on a shaker at 28°C for 2-3 hours before being plated onto solid LB medium (containing rifampicin (0.05 mg / mL) and spectinomycin (0.025 mg / mL, both filtered and sterilized). The transformed Agrobacterium was grown on the medium at 28°C for 2-3 days. Single colonies on the medium were picked for identification using the same primers and procedures as above.

[0052] The correctly identified strain was placed in an Erlenmeyer flask containing 200 mL of LB medium and incubated overnight at 28°C with shaking. Flowering wild-type Arabidopsis thaliana Col-0 was transformed using the inflorescence infection method. The specific procedure was as follows: A single colony of the correctly identified transformed Agrobacterium was placed in a shaking tube and gently shaken. The next day, the bacterial solution in the shaking tube was transferred to an Erlenmeyer flask containing 200 mL of liquid LB containing the corresponding antibiotic and shaken vigorously. After the bacterial solution became turbid, it was poured into a centrifuge bottle and centrifuged at 4000 rpm for 10 minutes at 25°C. The liquid LB was discarded, and the Agrobacterium was resuspended in 150 mL of 5% sucrose solution containing 0.02% Silwet L-77 using a pipette. The bacterial solution was then poured into a 100 mL graduated cylinder. Col-0 plants were inserted into the graduated cylinder and allowed to stand for 90 seconds before being removed, wrapped in plastic wrap to retain moisture, and placed in the dark for 12 hours before being placed in a normal culture room. The seeds of infected Arabidopsis thaliana are the T1 generation transgenic Arabidopsis thaliana. The T1 generation transgenic Arabidopsis thaliana seeds are sown in substrate soil, covered with a transparent lid, and exposed to light for 5-7 days to germinate. After the seedlings develop two cotyledons, a 1:1000 dilution of glufosinate-ammonium solution is sprayed to screen for positive seedlings. The seedlings that survive normally are the pFRK1::GUS transgenic Arabidopsis thaliana positive seedlings, and the seeds from these positive seedlings can be harvested for use.

[0053] 2. High-throughput screening of natural small molecules using biochemical methods.

[0054] The pFRK1::GUS transgenic Arabidopsis seeds were disinfected for 15 minutes using a commercially available 84 disinfectant solution (approximately 4% effective chlorine content):water = 3:7, and then rinsed 5-6 times with sterile water. After 2 days in a 4°C refrigerator, the seeds were used. A solid culture medium containing 2.25 g / L MS, 5 g / L sucrose, and 0.4% Phytagel (Shanghai Yuanye Biotechnology Co., Ltd.) was melted by heating, and 80 μL of this medium was added to each well of a 96-well shake plate. The disinfected Arabidopsis seeds were resuspended in 0.1% agarose gel (Nanjing Sangon Biotech Co., Ltd.), and then spotted onto 96-well transparent shake plates. The seeds were grown at 23°C for 5-7 days under 16h / 8h light / dark conditions. All sowing operations were performed in a clean bench, and the culture medium, 96-well plates, and pipette tips were all sterile. (The culture medium used for Arabidopsis thaliana growth will be referred to as 1 / 2MS solid medium in the following text, and the sterilization method is the same as here).

[0055] The synthesized PACAP powder standard (purity ≥95%) was dissolved in water to prepare a stock solution (10 mM). The stock solution was diluted to a working concentration gradient to form treatment groups (10 μM, 50 μM, 100 μM, etc.). Mock (dd H2O) and 100 nM flg22 were used as negative and positive controls, respectively. The 1-100 μM solutions of the control and treatment groups were added to the above-mentioned 96-well plates, ensuring that the seedlings in the wells were submerged. After 5 hours of treatment, the solution in the plate was aspirated with a pipette, and GUS staining buffer containing 1% X-Gluc (5-Bromo-4-chloro-3-indolylβ-D-glucuronide cyclohexylammonium salt) was added (preparation method is shown in Table 5).

[0056] Table 5 GUS staining buffer

[0057]

[0058] Then, stain at 37°C in the dark, observing every 2 hours. When the seedlings treated with flg22 (positive control) produce a distinct blue substance, the staining solution in the 96-well plate can be aspirated, and the leaves can be decolorized with 95% alcohol for further observation. If the Arabidopsis treated with the natural product produces the aforementioned blue color, it indicates that the natural product can activate the expression of the Arabidopsis immune marker gene FRK1, which has a certain function of activating plant immunity.

[0059] See results Figure 1The results showed that PACAP could induce blue staining in pFRK1-GUS transgenic Arabidopsis thaliana, and the staining degree became more obvious with increasing concentration, indicating that PACAP promotes the expression of the FRK1 gene in Arabidopsis thaliana.

[0060] Example 2: qRT-PCR detection of FRK1 gene and other immune-related genes expression

[0061] Col-0 seedlings were immersed in 25 μM PACAP 1-38 solution for 0, 2, 4, and 6 hours, respectively, with a 4-hour treatment with 100 nM flg22 as a positive control. Samples were then flash-frozen in liquid nitrogen. Total RNA was extracted from the plant material using the Kangwei Century Ultrapure RNA Kit. The RNA at the determined concentration was reverse transcribed using the Novizan HisScript III IstStrand cDNA Synthesis Kit (+gDNA wiper). qRT-PCR reactions were performed using the Novizan (ChamQ SYBR qPCRMaster Mix) quantitative kit. During the preparation process, it was important to avoid light exposure, as the premix contains SYBR Green I. The specific preparation system is shown in Table 6.

[0062] Table 6 RT-qPCR Configuration System

[0063]

[0064] The instrument used for qRT-PCR was the Biorad CFX Connect, and the specific reaction procedure is shown in Table 7.

[0065] Table 7 RT-qPCR Running Procedure

[0066]

[0067] qRT-PCR primers were designed based on the genome and cDNA sequences. The gene amplification primer sequences are shown in Table 8.

[0068] Table 8. Primer List for RT-qPCR

[0069]

[0070] Quantitative results of gene expression levels, such as Figure 2 As shown, PACAP promotes the expression of immune marker genes FRK1 and WRKY29, and the fold induction increases with the extension of treatment time.

[0071] Example 3: PACAP promotes calcium ion influx in Arabidopsis thaliana

[0072] This experiment used Col-Q Arabidopsis thaliana transgenic material, which stably expresses exogenously introduced aequorin protein. In the presence of oxygen and the substrate coelenterate (CTZ), they can form a luminescent complex with the aequorin protein. Calcium ions (Ca...) 2+ When combined with this complex, it can release carbon dioxide and produce blue fluorescence.

[0073] In this experiment, diluted MES (200 mM, 100×), CaCl2 (1 M, 100×), and CTZ mixture (1 mM, 100×) were first added to each well of a white 96-well plate. Then, 5-7 day old Arabidopsis Col-Q seedlings of uniform size (germinated from 15 cm culture dishes) were added to each well. After standing overnight, the next day, negative control DMSO, positive control flg22, and different concentrations of PACAP 1-38 were added to the 96 wells, and the chemiluminescence of Arabidopsis was detected using a microplate reader (Guangzhou Boluteng). The single reading time per well was 1 second. Since the calcium ion influx process was relatively rapid, it was advisable to test 3 columns (24 wells) of the 96-well plate each time, with 60 cycles (adjusted according to the total duration of approximately 20 minutes).

[0074] Calcium plays a crucial role in plant immune signaling as a key second messenger in all eukaryotic cells. Under normal physiological conditions, the concentration of free calcium in the apoplast is significantly higher than that in the cytosol. Pathogen infection or elicitor recognition triggers a rapid calcium influx, initiating downstream immune signaling. Figure 3 Chemiluminescence assays showed that PACAP 1-38 rapidly induced an increase in cytoplasmic calcium ion concentration in Arabidopsis thaliana in a dose-dependent manner. Notably, both PACAP 1-38 and PACAP 6-38 elicited significant calcium signaling, while PACAP 1-27 showed weaker activity compared to the other two variants.

[0075] Example 4: PACAP activation of Arabidopsis MAPKs

[0076] Arabidopsis thaliana Col-0 seedlings in good growth condition were removed from the culture medium and transferred to 12-well clear culture plates. The samples were soaked in sterile water overnight to maintain consistent sample condition. The next day, flg22 and PACAP were added to the 12-well plates to achieve their corresponding working concentrations. It is generally believed that MAPK activation of the PTI pathway is strongest within 15-30 minutes, while MAPK activation of the ETI pathway requires a longer time, typically several hours. Therefore, the treatment time gradients in this experiment included 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h. After the designated time, the Arabidopsis samples were removed, blotted dry with paper towels, placed in 1.5 mL centrifuge tubes, and small steel beads were added. The samples were then stored at -80°C.

[0077] The frozen sample was homogenized in a high-throughput tissue homogenizer. 100 µL of protein extraction buffer (150 mM NaCl, 1.0% Triton X-100, 50 mM Tris-HCl, pH 8.0) was added, mixed, and incubated on ice for 5 minutes. The sample was then centrifuged at 12,000 rpm for 10 minutes at 4°C. 80 µL of the supernatant was transferred to a new tube, and 20 µL of 5X Loading Buffer was added. The sample was incubated in a metal bath at 80°C for 10 minutes.

[0078] Assemble the electrophoresis tank, remove the pre-cast gel, peel off the membrane on the gel surface, remove the comb, insert the gel plate into the electrophoresis tank, and close the latch. Add running buffer to the electrophoresis tank, and load samples, 15 µL per well, and 3 µL per protein marker well. If there are blank wells, fill them with 1× loading buffer. Cover the electrophoresis tank and set the electrophoresis apparatus to low voltage (80V) for 2.5 h. Cut the target band (approximately 53 kDa) according to the size of the protein marker and the target protein, and immerse it in transfer buffer. Soak the cellulose acetate (PVDF) membrane in methanol for one minute, then immerse it in transfer buffer to fully moisten it. Stack the membranes in the following order: positive electrode - sponge - filter paper - PVDF membrane - protein gel - filter paper - sponge - negative electrode on the transfer apparatus, and squeeze out the water to remove air bubbles, keeping it moist. Connect the electrophoresis apparatus, set it to 100 V, and perform transfer for 1.5 h. After transfer, the PVDF membrane was blocked horizontally on a shaker at room temperature for 1-2 hours with TBST buffer containing 5% skim milk powder. Then, it was incubated with primary antibody (antiphospho-p44 / 42MPKs, 1:2000) at room temperature for 1-2 hours. After incubation, the PVDF membrane was washed three times with TBST buffer on a horizontal shaker for 15 minutes each time. The membrane was then transferred to a universal secondary antibody (peroxidase-conjugated universal rabbit IgG, 1:15000) and incubated. It was then washed three times with TBST for 15 minutes each time. The membrane was then imaged at 10 s, 30 s, 1 min, 5 min, and 15 min to analyze the phosphorylation of the corresponding proteins, selecting the results from the 5-minute exposure time. The PVDF membrane was stained with Ponceau S for 30 s to check the uniformity of total protein extraction in each sample.

[0079] Western blotting was used to detect phosphorylation of MAPK proteins in Arabidopsis seedlings. The results are as follows: Figure 4 As shown, PACAP1-38 and its truncated forms PACAP 6-38 and PACAP 1-27 can all activate the phosphorylation of Arabidopsis MAPK proteins, and this induction can be detected 15 minutes after treatment. PACAP1-38 or PACAP 6-38 showed stronger activity in activating MAPK phosphorylation, while PACAP1-27 showed relatively weaker activity. This indicates that PACAP activates a core pathway in plant immune signaling, and its mode of action is similar to that of PAMP molecules (such as flg22).

[0080] Example 5: PACAP enhances resistance of Arabidopsis thaliana to Pseudomonas syringae.

[0081] The pathogenic strain of *Pseudomonas syringae* pv. tomato DC3000 was cultured on King's B (KB) solid medium at 28 °C for 48 h. A single colony was then picked and inoculated into 4 mL of KB liquid medium and cultured overnight at 28 °C with shaking at 200 rpm until the OD of the bacterial culture was reached. 600 The value reached 0.6-0.8. After cultivation, the bacterial culture was centrifuged to collect the bacterial cells, and then resuspended in 10 mM MgCl2 to adjust the bacterial concentration to OD. 600 =0.002, used as the bacterial suspension for subsequent inoculation.

[0082] To test whether peptide pretreatment could enhance plant resistance, fully expanded leaves of 4-week-old Arabidopsis thaliana plants were selected. 24 hours before pathogen inoculation, the leaves were injected with 1 mL needleless syringe. The treatment solution consisted of different concentrations of PACAP1-38, PACAP1-27, or PACAP 6-38 (25 μM, 50 μM, and 100 μM). The control group was injected with sterile water.

[0083] After 24 h of pretreatment, the leaves were inoculated with the prepared P. syringae pv. tomato DC3000 bacterial suspension. After inoculation, excess bacterial suspension on the leaf surface was absorbed with sterile filter paper, and the plants were placed under high humidity growth conditions for cultivation.

[0084] On the second day after inoculation, three leaf discs with a diameter of 0.5–0.6 cm were taken from each leaf using a perforator. These discs were first surface-sterilized with 75% ethanol for 30 seconds, then rinsed twice with sterile distilled water, and subsequently homogenized in 10 mM MgCl2. The homogenate was serially diluted and spread onto KB solid medium containing 50 μg / mL rifampicin. After incubation at 28 ℃ for 48 h, colony-forming units (CFU) were counted. Results are as follows: Figure 5 As shown, PACAP1-38, PACAP1-27, or PACAP 6-38 all enhanced the resistance of Arabidopsis thaliana to the pathogen Pseudomonas syringae pv. tomato DC3000.

[0085] Example 6: PACAP enhances tobacco resistance to Ralstonia solanacearum.

[0086] To conduct a Ralstonia solanacearum infection test, tobacco seedlings (cultivar 'Yabuli', Nicotiana tabacum cv. Yabuli) that had been growing on 1 / 2 MS agar plates for two weeks were carefully removed and incubated overnight in sterile double-distilled water. First, the roots were lightly incised with a sterile blade. Then, the seedlings were infiltrated with different concentrations of PACAP1-38, PACAP1-27, PACAP 6–38, or a negative control, in double-distilled water. Twelve hours after treatment, inoculation was performed on the same tissue sites with a concentration of 1 × 10⁻⁶. 6 CFU mL -1 Ralstonia solanacearum suspension.

[0087] One day after inoculation, samples were taken for quantitative bacterial analysis: the tissue was weighed, homogenized in sterile water, serially diluted, and spread onto solid CPG medium. After incubation at 28°C for 2 days, colony forming units (CFUs) were counted, and the results were expressed as the number of CFUs per gram of fresh tissue weight.

[0088] Consistent with the *Pseudomonas syringae* infection experiments, PACAP 1-38, PACAP 1-27, and PACAP 6-38 all enhanced tobacco's resistance to *Ralstonia solanacearum*. This also indicates their broad role in plant resistance to bacterial diseases.

[0089] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. The application of a PACAP polypeptide in the preparation of a plant immune modulator, wherein the PACAP polypeptide has the activity of enhancing plant immune response.

2. The application according to claim 1, characterized in that, The PACAP polypeptide is selected from: the full-length PACAP1-38 amino acid sequence as shown in SEQ ID NO. 1, the PACAP1-27 fragment as shown in SEQ ID NO. 2, or the PACAP6-38 fragment as shown in SEQ ID NO.

3.

3. The application according to claim 1 or 2, characterized in that, The plant immunomodulator is used to activate the plant's immune response.

4. The application according to claim 3, characterized in that, The activated plant immune response manifests as one or more of the following: inducing the expression of immune marker genes FRK1 and WRKY29, promoting the phosphorylation of MAPK proteins, or triggering intracellular calcium ion influx.

5. The application according to claim 1 or 2, characterized in that, The plant immunomodulator is used to enhance the plant's resistance to bacterial pathogens.

6. The application according to claim 5, characterized in that, The bacterial pathogens are *Pseudomonas syringae* and *Ralstonia solanacearum*.

7. A plant immunomodulator comprising PACAP with an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, and an agronomically acceptable carrier and / or adjuvant.

8. The plant immunomodulator according to claim 7, characterized in that, The regulator is a wettable powder, aqueous solution, or emulsifiable concentrate.

9. A method for enhancing plant immune response, characterized in that, The step includes applying an effective amount of the plant immunomodulator of claim 7 or 8 to the plant or its growing environment.

10. A method for preventing and controlling plant diseases caused by *Pseudomonas syringae* and *Ralstonia solanacearum*, characterized in that, The method involves applying an effective amount of PACAP, with an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, to a plant, such as Arabidopsis thaliana or tobacco.

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