A plant immunity induction polypeptide derived from citrus huanglongbing bacterium and application thereof

Abstract

The application discloses a plant immune induction polypeptide derived from citrus Huanglongbing bacteria and application thereof, wherein the amino acid sequence of the polypeptide is selected from SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and the polypeptide also comprises a homologous variant with equivalent immune induction function, and the application also discloses a polynucleotide, a recombination carrier, an engineering strain and an agricultural composition containing the polypeptide, wherein the agricultural composition can be prepared into an aqueous agent, a powder agent or a granular agent, and is applied by spraying, root irrigation or tree trunk injection. Experiments prove that the polypeptide can induce a plant to produce a reactive oxygen burst, activate a MAPK cascade signal path and systematically up-regulate the expression of defense genes such as PR1. The polypeptide can significantly inhibit the colonization and proliferation of pathogenic bacteria such as Pseudomonas syringae in a plant body, has the advantages of high activity, strong specificity and green safety and the like, and provides a new strategy for the prevention and control of plant bacterial diseases.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a plant immune-inducing polypeptide derived from Huanglongbing fungus of citrus and its application. Background Technology

[0002] Plants face the threat of infection from various pathogenic microorganisms during their growth and development. To resist external biotic stress, plants have evolved innate immune defense mechanisms. Pattern recognition receptors on the cell membrane surface can recognize evolutionarily conserved molecular features in pathogenic microorganisms, namely pathogen-associated molecular patterns. This recognition triggers the plant's innate immune response, inducing physiological and biochemical changes including reactive oxygen species bursts, activation of mitogen-activated protein kinase cascade pathways, and upregulation of defense gene expression, thereby limiting pathogen invasion in the early stages of infection.

[0003] In agricultural production, chemical control is the primary measure for controlling bacterial plant diseases. However, the long-term and extensive use of chemical pesticides has led to negative consequences such as environmental residues, food safety risks, and increased pathogen resistance. Inducing plants to activate their own immune systems using pathogen-associated molecular models is considered an environmentally friendly disease control strategy. While some plant immune inducers, such as bacterial flagellin or elongation factors, have been used in basic research or agricultural applications, the development of highly active and widely applicable bio-based immune inducers remains a research hotspot in this field.

[0004] Despite the significant advantages of biomolecular-based immune induction strategies, existing plant immune induction technologies still have limitations. For fastidious pathogens such as *Hydrocotyledon citrus*, which are difficult to culture in vitro, there is currently a lack of efficient and stable pathogen-associated molecular models derived from these pathogens. Existing technologies lack novel bio-derived molecules capable of rapidly and strongly activating the plant immune system (e.g., rapidly inducing reactive oxygen species bursts and kinase pathway phosphorylation). This results in a lack of highly specific and inducing biological control methods for such diseases, limiting the development and application of green control technologies for these diseases. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a plant immune-inducing polypeptide derived from Huanglongbing fungus of citrus and its application, addressing the problem of the lack of highly efficient, low-toxicity biological control agents capable of inducing systemic resistance to bacterial plant diseases in existing technologies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In its first aspect, the present invention provides a plant immune-inducing polypeptide. The amino acid sequence of the plant immune-inducing polypeptide is SEQ ID NO:1. Furthermore, the present invention also includes homologous variants of the above sequence. The homologous variant refers to an amino acid sequence that, based on the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, undergoes a conserved substitution, deletion, or addition of one to three amino acid residues, and is capable of inducing the production of reactive oxygen species (ROS) or activating the mitogen-activated protein kinase pathway in plants; or an amino acid sequence that shares at least 85% identity with the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and is capable of inducing the production of ROS or activating the mitogen-activated protein kinase pathway in plants.

[0007] A second aspect of the present invention provides a polynucleotide encoding the above-mentioned plant immune-inducing polypeptide. The nucleotide sequence of the polynucleotide is SEQ ID NO:4; or it is a polynucleotide encoding the above-mentioned homologous variant amino acid sequence. The SEQ ID NO:4 sequence has been optimized with plant codons for efficient expression in plant cells.

[0008] A third aspect of the present invention provides a recombinant vector comprising the aforementioned polynucleotide. The recombinant vector is pCNF3, and the polynucleotide is inserted between the CaMV 35S promoter and the NOS terminator of the recombinant vector.

[0009] A fourth aspect of the present invention provides an engineered bacterial strain comprising the above-described recombinant vector. The engineered bacterial strain is *Agrobacterium tumefaciens* (…). Agrobacterium tumefaciens Specifically, it refers to strain GV3101.

[0010] A fifth aspect of the present invention provides an agricultural composition. The agricultural composition comprises an effective amount of the aforementioned plant immune-inducing polypeptide as an active ingredient, and a pesticide-acceptable carrier or adjuvant. The formulation of the agricultural composition includes an aqueous solution, a wettable powder, or a slow-release granule. As a preferred embodiment: the aqueous solution comprises the plant immune-inducing polypeptide, polyoxyethylene sorbitan monolaurate as a wetting agent, disodium EDTA as a stabilizer, sodium benzoate as a preservative, and deionized water; the wettable powder comprises the plant immune-inducing polypeptide, sodium lignosulfonate as a dispersant, sodium dodecyl sulfate as a wetting agent, and kaolin or diatomaceous earth as a carrier; the slow-release granule comprises the plant immune-inducing polypeptide, sodium carboxymethyl cellulose as a binder, bentonite as a disintegrant, and fine sand as a carrier.

[0011] A sixth aspect of the present invention provides the use of the above-mentioned plant immune-inducing polypeptides, polynucleotides, recombinant vectors, engineered strains, or agricultural compositions in the preparation of formulations for the prevention and control of bacterial plant diseases, wherein the bacterial plant diseases are: spp. of phloem ( Candidatus Liberibacter )include Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, Candidatus Liberibacter American Citrus Huanglongbing caused by, or by Candidatus Liberibacter solanacearum Diseases caused by; Pseudomonas spp. ( Pseudomonas ), including Pseudomonas syringae, which causes leaf spot or canker. Xanthomonas ( ) Xanthomonas ), including those that cause citrus canker. Xanthomonas citrisbsp. lemons .

[0012] A seventh aspect of the present invention provides a method for controlling bacterial diseases of plants. The method includes applying the above-described agricultural composition to plants; the application method is selected from foliar spraying, root irrigation, root application, or trunk injection. As a preferred embodiment: in the foliar spraying, the agricultural composition is diluted to a final concentration of 1 to 10 micromoles / liter for the plant immune-inducing polypeptide; in the trunk injection, a hole is drilled downwards at a 45-degree angle at a distance of 20 to 30 centimeters from the ground on the plant trunk using an electric drill, the hole penetrating the phloem and entering the xylem 1 to 2 centimeters, and then an aqueous solution of the agricultural composition is injected.

[0013] This invention provides a plant immune-inducing polypeptide derived from *Huanglongbing* fungus of citrus and its application. It possesses the following beneficial effects: 1. This invention induces a burst of reactive oxygen species from chloroplasts in plant leaves within hours by applying a micromolar level polypeptide solution, and simultaneously promotes the phosphorylation modification of mitogen-activated protein kinases MPK3 and MPK6, thereby activating the intracellular immune signal transduction cascade pathway and achieving the technical effect of rapidly initiating the plant's innate immune response and establishing an early defense barrier.

[0014] 2. This invention, through the exogenous application of this polypeptide molecule, can systematically regulate the transcriptional expression of defense-related genes in plants, significantly upregulating the expression levels of salicylic acid signaling pathway gene PR1, transcription factor WRKY, and oxidase gene RBOHB. This molecular-level defense mobilization enables plants to significantly inhibit the colonization and reproduction of pathogens such as Pseudomonas syringae in plant tissues when facing infection, greatly reducing the number of colonies in leaves and alleviating tissue necrosis symptoms, thus achieving the technical effect of highly efficient prevention and control of bacterial diseases in plants.

[0015] 3. This invention limits the spread of pathogens by mobilizing the plant's endogenous immune system, which not only effectively reduces the risk of pathogens developing drug resistance, but also eliminates pesticide residues and toxic threats to non-target organisms because the polypeptides are derived from natural organisms and are easily degraded in the environment, thus achieving a synergistic unity between green disease control and ecological environmental safety.

[0016] 4. The polypeptides and their potent homologs provided by this invention retain specific structural features that enable plants to recognize them efficiently. By applying these polypeptides exogenously, the natural recognition defects of plants against pathogens can be effectively remedied, and the immune system that has been evaded by pathogens can be forcibly activated, providing a novel molecular strategy for solving the problem of pathogen immune escape. Attached Figure Description

[0017] Figure 1 This is a diagram showing the RT-qPCR detection results of the plant immune-inducing polypeptide (Pal19 / SEQ ID NO:1) of the present invention inducing the expression of plant defense-related genes; Figure 2 This is a Western blot analysis result of the plant immune-inducing peptide activating the MAPK cascade signaling pathway in this invention. Figure 3 This is a microscopic image of the detection of reactive oxygen species (ROS) bursts in plant leaves using the H2DCF-DA fluorescent probe of this invention. Figure 4 This is a graph showing the in vivo control effect of the plant immune-inducing peptide of this invention on Pseudomonas syringae (Pst DC3000). Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Please see the appendix Figure 1 This invention provides a plant immune-inducing polypeptide derived from *Citrus Huanglongbing* and its application. The implementation process includes the following steps (in the accompanying drawings and experimental descriptions of this invention, the code 'Pal19' refers to the plant immune-inducing polypeptide (SEQ ID NO:1) described in this invention): S100: Screening and Chemical Synthesis of Plant Immune-Inducing Peptides Using bioinformatics analysis methods, the pathogens causing citrus Huanglongbing (HLB) were compared. Ca. Liberibacter asiaticusGenomic data were used to screen out a highly conserved amino acid sequence in pathogens that can be recognized by plants, namely SEQ ID NO:1.

[0020] The target peptide was prepared using the Fmoc solid-phase peptide synthesis process. The synthesis process used resin as a solid-phase carrier, adding amino acid residues sequentially from the C-terminus to the N-terminus, and extending the peptide chain through deprotection, coupling, and cleavage reactions. The synthesized product was purified by high-performance liquid chromatography and analyzed by mass spectrometry, verifying that its molecular weight was consistent with the theoretical value and its purity was greater than 95%, thus eliminating the interference of impurities on subsequent bioactivity assays.

[0021] To clarify the scope of protection of this invention, the polypeptide prepared in this step is not limited to SEQ ID NO:1, but also includes homologous variants with equivalent immunoinducing functions. Homologous variants refer to amino acid sequences that, based on the SEQ ID NO:1 sequence, undergo conserved substitution, deletion, or addition of one to three amino acid residues, or have a sequence identity of SEQ ID NO:1 of 85% or higher, and are capable of inducing the production of reactive oxygen species or activating the mitogen-activated protein kinase pathway in plants.

[0022] S200: Detection of early immune response in plants The synthesized plant immune-inducing peptide was formulated into a solution with a concentration ranging from 1 µM to 10 µM (preferably 1 µM) and then contacted with plant leaf tissue. This step was used to verify the binding ability of the peptide molecules to pattern recognition receptors on the surface of plant cell membranes.

[0023] The reactive oxygen species (ROS) level in plant leaves was monitored using luminol chemiluminescence. If plant cells recognize a polypeptide signal, NADPH oxidases (such as RBOHD) on the cell membrane are phosphorylated and activated, releasing ROS into the extracellular space. The change in photon count (RLU) was continuously recorded from 0 to 60 minutes or 4 hours after treatment using a chemiluminescence analyzer, and the intensity of the immune response was quantified by the emission peak and integrated area.

[0024] S300: Phosphorylation analysis of intracellular signal transduction cascade pathways After detecting reactive oxygen species signals, the activation status of the mitogen-activated protein kinase (MAPK) cascade pathway was further analyzed by immunoblotting experiments.

[0025] Plant leaves treated with peptides at different time points (e.g., 15 minutes, 30 minutes, and 60 minutes) were collected, and total protein was extracted from the leaves. Hybridization and colorimetric analysis were performed using antibodies that specifically recognize phosphorylated threonine / tyrosine motifs. Phosphorylation band signals of MPK6 protein (around 43 kDa) and MPK3 protein (around 46 kDa) were observed. A significant enhancement of the band signal compared to the untreated control group indicated that the peptide-induced immune signal had been transmitted into the cell via a phosphorylation cascade.

[0026] S400: Quantitative assessment of transcriptional levels of plant defense genes: We used real-time quantitative PCR to detect changes in the expression of defense genes caused by signal transduction to the cell nucleus.

[0027] The study included the salicylic acid pathway marker gene PR1, the transcription factor WRKY, and the respiratory burst oxidase homolog gene RBOHB. Specific primers targeting these genes were designed, and plant housekeeping genes (such as Actin or EF1α) were used as internal controls. The relative gene expression levels in the peptide-treated group compared to the control group were calculated. The significant upregulation of gene expression levels confirmed the systematic activation of the plant's innate immune system at the transcriptional level.

[0028] S500: In vivo efficacy test against bacterial diseases Based on molecular-level verification, using *Pseudomonas syringae* (… P. syringae The test strain was used to evaluate the protective effect of the polypeptide on live plants.

[0029] A preventative treatment approach was adopted, in which immune-inducing peptides were sprayed or injected onto plant leaves 24 hours before inoculation with the pathogen. After the plants established an immune state, inoculation was carried out. After 3 to 5 days of inoculation and culture, leaf samples were collected, ground, diluted, and colony-forming units (CFU) were counted using the plate spread method.

[0030] The formula for calculating the antibacterial rate is as follows:

[0031] In the formula: Defined as antibacterial rate; Defined as the number of pathogen colonies per unit weight of leaves in the control group; Defined as the number of pathogen colonies per unit weight of leaves in the polypeptide-treated group.

[0032] Statistical analysis requires confirmation Significantly lower than And antibacterial rate The result was 90% or higher, which was used to confirm that the polypeptide could inhibit the colonization and proliferation of pathogens in plant tissues.

[0033] S600: Formulation and Application of Agricultural Preparations Combining bioactive peptides with pesticide adjuvants.

[0034] When preparing aqueous solutions, wetting surfactants (such as Tween-20) are added to reduce the surface tension of the solution. When preparing powders or granules, kaolin or diatomaceous earth is used as an inert carrier to adsorb polypeptide molecules. Depending on the crop growth characteristics, the formulation is applied by foliar spraying, root irrigation, or stem injection, so that the polypeptide molecules come into contact with plant tissues and induce systemic resistance, thereby preventing bacterial diseases.

[0035] To ensure that the technical solutions of the present invention can be fully reproduced by those skilled in the art, the general experimental materials, culture conditions and basic construction methods involved in each embodiment are described in detail below.

[0036] I. Cultivation of the test plant materials: The experiment used Tobacco Bunsen ( Nicotiana benthamiana As a model plant, sweet orange ( Citrus Chinese As a validation crop, seeds were surface-sterilized and sown in a substrate containing sterilized vermiculite and nutrient soil (volume ratio 1:1). The growing environment was controlled as follows: temperature 24±2℃, relative humidity 60%-70%, photoperiod 16h / 8h (light / dark), and light intensity... Plants that have grown to 4-5 weeks old and have healthy, undamaged leaves were selected for subsequent experiments.

[0037] II. Activation conditions for the tested strains: Selected *Pseudomonas syringae* tomato pathogenic strain ( P. syringae pv. Tomato DC3000 was used as the challenge target, and the strain was tested at a concentration of 35... On King's B (KB) medium containing rifampicin, cultured at 28°C until the logarithmic growth phase (… ), use sterile 10 before inoculation Resuspend the bacterial cells to the desired concentration.

[0038] III. Design Strategies for Peptides and Homologous Variants: The polypeptide sequences involved in this invention (including SEQ ID NO:1 and its variants) are all prepared by solid-phase synthesis and have a purity >95%.

[0039] Core sequence: SEQ ID NO:1 (derived from citrus Huanglongbing fungus) Candidatus Liberibacter Asian The sequence is: VYGHDEDAYAKNRRAIVFL.

[0040] Homologous variants: including variants with 1-3 conserved amino acid substitutions (such as R12K) based on SEQ ID NO:1, and variants with >85% sequence identity.

[0041] Control drug: flg22 polypeptide.

[0042] All peptides were formulated to 1 Store the mother liquor at -20°C.

[0043] IV. Fundamentals of Encoding Polynucleotides and Vector Construction: For the amino acid sequence of SEQ ID NO:1, the corresponding DNA coding sequence is synthesized according to the plant codon preference and cloned into the downstream of the strong promoter (such as CaMV 35S) of the plant binary expression vector (such as pCNF3). The constructed product is transformed into Agrobacterium GV3101 for transient expression or transgenic verification.

[0044] V. Agricultural Composite Additive System: Excipient systems used in formulation development include: Carriers: water, PBS buffer (liquid formulation); kaolin, diatomaceous earth (solid formulation).

[0045] Additives: Nonionic surfactants (such as Tween-20, Silwet L-77), typically used at concentrations ranging from 0.01% to 0.05% (v / v), to enhance wetting and spreading properties.

[0046] VI. Main Biochemical Testing Reagents ROS assay used luminol and horseradish peroxidase (HRP); Western blotting used antiphosphorylated MAPK (Erk1 / 2) specific antibody; qPCR used specific primers against PR1, WRKY, RBOHB and internal control Actin.

[0047] This embodiment details the process of obtaining the plant immune-inducing polypeptide and its homologs described in this invention. Bioinformatics design, chemical synthesis, and physicochemical property identification confirmed the material basis for subsequent bioactivity verification.

[0048] To verify the conservation and functional universality of the plant immune-inducing polypeptide described in this invention across different microbial sources, and to determine the technical protection boundaries of this invention, this embodiment selects two naturally occurring homologous variant sequences for comparative verification: SEQ ID NO:2 (derived from Escherichia coli) Escherichia coli The sequence is VLGHDEAAYSKNRRAVLVY. This sequence is highly similar to SEQ ID NO:1 in the core region, especially retaining the key NRRA domain, which is used to verify the functional conservation of this polypeptide family in different bacterial classes.

[0049] SEQ ID NO:3 (derived from Agrobacterium tumefaciens) A. tumefaciensThe sequence is VAVCDDISCWSQNRRAVTVL. Although there is significant variation in the N-terminal region, the conserved C-terminal motif is still preserved, which is used to verify the decisive role of the core motif in immune induction activity.

[0050] Furthermore, sequence alignment revealed that the tested pathogen used in this embodiment—*Pseudomonas syringae* (… P. syringes pv . A homologous sequence (VATGNDEQSWAQNRRVELRK) also exists in the tomato DC3000. Notably, compared with the highly active SEQ ID NO:1, the natural sequence of DC3000 shows significant differences at position 12 and the C-terminal region (such as replacing the basic lysine K with glutamine Q). This natural variation may be an "immune escape" feature evolved by the pathogen to evade plant immune recognition.

[0051] The specific sequence information has been updated as follows: SEQ ID NO:1:VYGHDEDAYAKNRRAIVFL SEQ ID NO:2:VLGHDEAAYSKNRRAVLVY SEQ ID NO:3:VAVCCDDISCWSQNRRAVTVL S112: Solid-phase peptide synthesis process The three peptides described above were prepared using the Fmoc solid-phase synthesis method. The synthesis process was completed on a multi-channel fully automated peptide synthesizer.

[0052] A resin pre-loaded with leucine, the first amino acid at the C-terminus, was selected as the solid-phase support. The degree of substitution of the resin was 0.5 mmol / g. The specific synthesis steps are as follows: Deprotection: Add a solution of N,N-dimethylformamide (DMF) containing 20% ​​(v / v) piperidine to the reactor and react for 15 minutes to remove the Fmoc protecting groups of amino acids on the resin and expose the free amino groups.

[0053] Washing: The resin was washed four times with DMF solvent to remove residual piperidine and reaction byproducts.

[0054] Coupling: Four molar equivalents of Fmoc-protected amino acids, activator HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate), and organic base DIEA (N,N-diisopropylethylamine) were dissolved in DMF, activated, and then added to the reaction column. The reaction was carried out at room temperature for 45 minutes. The amino acid linkage sequence strictly followed the sequence in S111 from the C-terminus to the N-terminus.

[0055] Capping: After each coupling step, unreacted free amino groups are capped by acetylation with a mixture of acetic anhydride and pyridine to block the elongation of short peptide byproducts.

[0056] After the complete sequence assembly was completed, the resin was treated with a cleavage reagent (95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane) at room temperature for 3 hours to cleave the peptide from the resin and simultaneously remove the side chain protecting groups. The filtrate was precipitated with cold diethyl ether, collected by centrifugation, and vacuum dried to obtain the crude peptide.

[0057] S113: Purification and Physicochemical Property Identification of Polypeptides The crude peptide was dissolved in a 10% acetonitrile aqueous solution, filtered through a 0.22-micron filter membrane, and purified using preparative high-performance liquid chromatography (Prep-HPLC). The purification conditions were set as follows: column: C18 reversed-phase preparative column (5-micron packing material, 20 mm inner diameter x 250 mm column length); mobile phase A: ultrapure water containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile containing 0.1% trifluoroacetic acid; elution program: flow rate 10 mL / min, with the proportion of mobile phase B increasing linearly from 25% to 55% within 25 minutes; detection wavelength: 220 nm.

[0058] The main peak fraction was collected and re-analyzed by analytical HPLC, confirming that the chromatographic purity of SEQ ID NO:1 to SEQ ID NO:3 was greater than or equal to 95.0%.

[0059] The precise molecular weight of the purified product was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry to confirm the correctness of the synthetic sequence. The identification results are as follows: SEQ ID NO:1: The theoretical molecular weight of the single isotope is 2234.14 Da, and the measured peak value of [M+H]+ is 2235.2 Da (remaining unchanged).

[0060] SEQ ID NO:2: The molecular formula is C94H152N26O28, the theoretical molecular weight is about 2126.4 Da, and the measured peak value of [M+H]+ is 2127.5 Da.

[0061] SEQ ID NO:3: The molecular formula is C89H142N26O27S2, the theoretical molecular weight is about 2164.5 Da, and the measured peak value of [M+H]+ is 2165.6 Da.

[0062] Mass spectrometry analysis showed that the measured molecular weight of the synthesized product deviated from the theoretical calculation within a reasonable range, confirming the successful acquisition of the target sequence peptide. The prepared peptide was freeze-dried into a white powder, aliquoted, and stored in a -20°C drying environment.

[0063] By constructing a recombinant vector containing the coding sequences of SEQ ID NO:1 and its homologous variants, technical support is provided for verifying the endogenous expression of peptides in plants and their disease resistance function.

[0064] S210: Optimization and Synthesis of Encoding Polynucleotide Sequences Given the differences in codon usage preferences between prokaryotes and eukaryotes, codon optimization was performed on the coding sequence to improve the translation efficiency of peptides derived from Huanglongbing fungus in plant cells.

[0065] Using the amino acid sequence of SEQ ID NO:1 (VYGHDEDAYAKNRRAIVFL) as a template, and referring to the sample from *Nicotiana Bunsenius* (… N. Bentham's The codon frequency table is used to convert the amino acid sequence into a DNA sequence. During optimization, low-abundance codons are replaced with codons preferred by highly expressed genes in plants, and the GC content is adjusted to between 45% and 55%. At the same time, consecutive adenine (A) or thymine (T) sequences are avoided to prevent premature termination of transcription.

[0066] The optimized nucleotide sequence is named SEQ ID NO:4, and its specific sequence is as follows: 5'-GTG TAC GGC CAC GAC GAG GAC GCC TAC GCC AAG AAC AGG AGG GCC ATCGTG TTC CTG-3' To accommodate subsequent vector construction, a BamHI restriction site (GGATCC) and a Kozak sequence (containing the start codon ATG) were added to the 5' end of SEQ ID NO:4, and a stop codon (TAA) and a SalI restriction site (GTCGAC) were added to the 3' end. The full-length DNA fragment and its complementary strand were synthesized by a biotechnology company and cloned into the pUC57 vector. Sequencing confirmed that the sequence was correct.

[0067] For the homologous variant SEQ ID NO:2 (a sequence derived from E. coli) selected in Example 1, its encoding nucleotide sequence was designed using the same strategy. Based on the codon preference of Tobacco Bunsen, the corresponding DNA fragment was artificially synthesized to construct the variant encoding sequence for subsequent comparative experiments.

[0068] S220: Construction of a plant binary expression vector pCNF3 was selected as the backbone of the plant expression vector, which contains the cauliflower mosaic virus CaMV 35S promoter and the cauliflower broccoli synthase (NOS) terminator.

[0069] The construction steps are as follows: S221: Double enzyme digestion reaction. Extract the pUC57 plasmid containing the target gene and the pCNF3 empty vector plasmid separately. Add plasmid DNA, BamHI restriction endonuclease, SalI restriction endonuclease and corresponding buffer to a 50 μL reaction system and incubate at 37 degrees Celsius for 2 hours.

[0070] S222: Product purification. The enzyme digestion products were separated by 1% agarose gel electrophoresis. Approximately 100 bp of the target gene fragment and approximately 10 kb of the vector backbone were excised, and the DNA was recovered using a silica gel membrane centrifugation column method.

[0071] S223: Ligation reaction. The recovered target gene fragment is mixed with the linearized vector at a molar ratio of 5:1, T4 DNA ligase is added, and ligation is carried out at 16 degrees Celsius for 12 hours.

[0072] S224: Transformation and Identification. The ligation product was transformed into *E. coli* DH5α competent cells. Positive clones were screened on LB agar plates containing 50 mg / L kanamycin. Single colonies were picked for PCR identification and then sequenced for verification. Sequencing results showed that the target gene was correctly inserted between the promoter and terminator, with no frame shift.

[0073] The recombinant plasmid that was constructed was named pCNF3-35S- C LasPAMP1.

[0074] Using the same procedure, the SEQ ID NO:2 variant coding sequence prepared in S210 was constructed into the pCNF3 vector and named pCNF3-35S-CLasPAMP1-var for subsequent comparative experiments.

[0075] S230: Preparation of Agrobacterium engineered strains The recombinant plasmid constructed from S220 was introduced into Agrobacterium tumefaciens ( A. tumefaciens ) GV3101 strain.

[0076] The operation steps are as follows: S231: Transformation. Thaw 100 μL of GV3101 competent cells on ice, add 1 μg of recombinant plasmid, mix gently, and incubate on ice for 30 minutes. Quick-freeze in liquid nitrogen for 5 minutes, followed by heat shock in a 37°C water bath for 5 minutes.

[0077] S232: Resuscitation: Add 800 μL of antibiotic-free LB liquid medium and incubate at 28°C and 200 rpm for 3 hours with shaking.

[0078] S233: Screening, centrifugation to collect bacterial cells, resuspending and plating on a medium containing 50... g / L kanamycin, 35 g / L rifampin and 25 On LB solid plates containing g / L gentamicin.

[0079] S234: Validation. After incubating upside down at 28°C for 48 hours, a single colony was picked for colony PCR validation. Electrophoresis results showed a specific band of approximately 100 bp, confirming that the recombinant plasmid had been successfully transformed into Agrobacterium.

[0080] This embodiment verifies the ability of SEQ ID NO:1 and its homologs, as pathogen-associated molecular patterns (PAMPs), to bind to receptors on plant cell surfaces and trigger early immune responses. Reactive oxygen species (ROS) bursts are an early biochemical indicator of the activation of the plant's innate immune system; the rate and intensity of ROS production reflect the activity level of immune inducers.

[0081] S310: Preparation and Restoration of Plant Materials Four-week-old tobacco plants cultivated in Example 1.2 were selected. N. benthamiana Select a healthy leaf that is fully expanded in the middle of the plant, and use a 4 mm diameter punch to punch out leaf discs, avoiding the veins.

[0082] The obtained leaf discs were placed in sterile deionized water and allowed to float and stand at room temperature for 12 to 16 hours. This settling process was used to eliminate the release of background reactive oxygen species caused by mechanical perforation damage, ensuring that the subsequently detected signal originated from peptide induction and eliminating false positive results.

[0083] S320: Establishment and processing of the detection system The experiment was set up with 5 treatment groups, namely: SEQ ID NO:1 treatment group: containing 1 μmol / L ( )Detection solution for SEQ ID NO:1 peptide; SEQ ID NO:2 variant treatment group: containing 1 μmol / L ( The detection solution for SEQ ID NO:2 peptide was used to verify the functional retention after conserved amino acid substitution. SEQ ID NO:3 variant treatment group: containing 1 μmol / L ( The detection solution for SEQ ID NO:3 peptide was used to verify the functional retention of highly homologous sequences. Positive control group: containing 1 μmol / L ( ) detection solution for flg22 peptide; Negative control group: an equal volume of sterile deionized water.

[0084] The basic formula of the test solution is: containing 34 micrograms / mL ( ) luminol and 20 micrograms / ml ( The above-mentioned treatment solution was prepared by dissolving each group of polypeptides in a sterile aqueous solution of horseradish peroxidase (HRP) in the basic formula.

[0085] To conduct cellular localization observation of reactive oxygen species, an additional set of leaf discs was prepared for fluorescent probe detection. The fluorescent probe used was 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), prepared as a working solution at 10 μmol / L.

[0086] S330: Real-time monitoring of chemiluminescence signals Transfer the recovered leaf discs from S310 to white 96-well microplates, one disc per well, with eight biological replicates per group. Remove excess water from the wells and add 100 μL of the corresponding treatment group detection solution prepared in S320 to each well.

[0087] Immediately place the ELISA plate in a multi-functional microplate reader for detection. Set the detection parameters as follows: chemiluminescence mode, integration time 1000 ms, detection interval 2 minutes, and continuous detection time 60 minutes. Record the relative luminescence units at each time point.

[0088] Further, fluorescence microscopy observation of reactive oxygen species was performed by immersing leaf discs treated with peptides for 30 minutes in H2DCF-DA probe solution prepared by S320 and incubating them in the dark for 10 to 20 minutes. The leaves were then removed and rinsed three times with sterile water to remove excess dye. They were then observed under a laser confocal microscope with the excitation wavelength set to 488 nm and the emission wavelength set to 525 nm.

[0089] S340: Analysis of Immune Induction Results Export time-luminescence intensity data and plot kinetic curves. Detection results show: Response time: The RLU value was detected to increase 2 to 4 minutes after the addition of SEQ ID NO:1.

[0090] Peak characteristics: The luminescence intensity reached its peak 15 to 20 minutes after treatment. The peak intensity of the ROS burst induced by SEQ ID NO:1 reached more than 85% of that of the positive control flg22 treatment group.

[0091] Variant activity: Both SEQ ID NO:2 (R13K substitution) and SEQ ID NO:3 (V1I substitution) treatments induced single-peak ROS burst curves. The peak intensity of SEQ ID NO:2 was 80% of that of SEQ ID NO:1, and the peak intensity of SEQ ID NO:3 was 95% of that of SEQ ID NO:1.

[0092] Experimental data confirm that homologous variants retain the biological functions of recognizing receptors and triggering immune responses after undergoing conserved changes in their amino acid sequences.

[0093] Control results: The negative control group maintained the baseline level of RLU value throughout the testing period.

[0094] Microscopic imaging results: such as Figure 3 As shown, the negative control group (MOCK) only showed weak background fluorescence; while the leaf intercellular spaces and cell edges of the SEQ ID NO:1 treatment group (marked as Pal19 in the figure) and the positive control group (flg22) showed high-intensity green fluorescence signals, which directly confirmed that the peptide treatment induced a large amount of reactive oxygen species.

[0095] S350: Long-term induction monitoring Take another set of samples and extend the testing time to 4 hours.

[0096] The results showed that although the RLU value of the SEQ ID NO:1 treatment group showed a decreasing trend after 30 minutes, it was still higher than the baseline of the negative control group in the following 3.5 hours, which is consistent with the description in the briefing materials regarding the induction of immune response within 4 hours.

[0097] This embodiment uses Western blotting to verify the activation effect of SEQ ID NO:1 and its homologs on the plant mitogen-activated protein kinase (MAPK) cascade pathway at the protein level. The phosphorylation levels of MPK3 and MPK6 were detected to evaluate intracellular immune signal transduction.

[0098] S410: Plant Material Processing and Sample Collection Four-week-old Tobacco Bunsenior and one-year-old sweet orange seedlings of uniform growth were selected, and a sterile aqueous solution containing 1 μmol / L of SEQ ID NO:1 polypeptide was prepared as the treatment solution. The treatment solution was injected into the mesophyll tissue from the back of the leaf using a needleless syringe, ensuring that the liquid completely saturated the injection area.

[0099] Leaf samples were collected at 0, 15, 30, and 60 minutes after treatment. The sample collected at 0 minutes served as an untreated control. A sample containing 1 μmol / L (…) was also collected. For the treatment groups of SEQ ID NO:2 variant and SEQ ID NO:3 variant, samples were collected 15 minutes after treatment. The collected leaves were immediately flash-frozen in liquid nitrogen and then stored in a freezer at -80 degrees Celsius.

[0100] S420: Total Protein Extraction The frozen leaf samples were ground into powder in liquid nitrogen. Two volumes (w / v) of protein extraction buffer were added to the powder.

[0101] The formulation of the extraction buffer is as follows: 50 mmol / L ( Tris-HCl (pH 7.5); 150 mmol / L ( Sodium chloride; 10 mmol / L ( Magnesium chloride; 1 millimole / liter ( EDTA-1,5-diaminetetraacetic acid; 10% glycerin; 1% Triton X-100; 1 millimole / liter ( ) Benzyl sulfonyl fluoride; 10 mmol / L ( Sodium fluoride (phosphatase inhibitor); 1 millimole / liter ( Sodium orthovanadate (phosphatase inhibitor).

[0102] Sodium fluoride and sodium orthovanadate were added to inhibit endogenous phosphatase activity and maintain the phosphorylation state of MPK3 and MPK6. Samples were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using the Bradford method, and proteins were denatured by boiling for 10 minutes with SDS loading buffer.

[0103] S430: SDS-PAGE electrophoresis and membrane transfer Prepare a 10% sodium dodecyl sulfate-polyacrylamide gel, with a sample loading of 20 micrograms of total protein per lane. Perform stacking gel electrophoresis at 80 volts and separating gel electrophoresis at 120 volts until the bromophenol blue indicator migrates to the bottom of the gel.

[0104] After electrophoresis, the proteins on the gel were transferred to a polyvinylidene fluoride membrane using a wet transfer method. The transfer conditions were: constant current of 250 mA for 90 minutes, with the buffer solution kept at a low temperature (ice bath) during the transfer.

[0105] S440: Immunoblotting hybridization After transfer, the PVDF membrane was blocked at room temperature for 1 hour using TBST buffer containing 5% BSA.

[0106] Primary antibody incubation: Place the PVDF membrane in blocking buffer containing anti-phosphorylated MAPK antibody (Anti-Phospho-p44 / 42 MAPKErk1 / 2,Thr202 / Tyr204) at a dilution ratio of 1:2000 and incubate overnight at 4 degrees Celsius.

[0107] Wash the membrane: Wash the PVDF membrane three times with TBST buffer, 10 minutes each time.

[0108] Secondary antibody incubation: Place the PVDF membrane in a solution of goat anti-rabbit IgG secondary antibody labeled with horseradish peroxidase (HRP) at a dilution ratio of 1:5000 and incubate at room temperature for 1 hour.

[0109] Color development: After washing the film 3 times, immerse the PVDF film in chemiluminescent substrate (ECL) for 1 minute and expose it using a chemiluminescent imaging system.

[0110] Internal control detection: The same PVDF membrane was treated with acidic glycine stripping buffer (pH 2.5) and then retested with anti-Actin antibody (dilution ratio 1:5000) as an internal control protein.

[0111] S450: Analysis of Signal Pathway Activation Results The exposure results showed that specific bands appeared near 43 kilodaltons (kDa) and 46 kilodaltons (kDa), corresponding to phosphorylated MPK6 (pMPK6) and phosphorylated MPK3 (pMPK3), respectively.

[0112] Kinetic analysis shows that: No obvious phosphorylation bands were detected in the 0-minute control group.

[0113] In the SEQ ID NO:1 treatment group, the band signals of pMPK3 and pMPK6 were significantly enhanced 15 minutes after treatment, reached a peak signal intensity at 30 minutes, and weakened at 60 minutes.

[0114] The SEQ ID NO:2 and SEQ ID NO:3 processing groups detected pMPK3 and pMPK6 bands at 15 minutes, with signal strengths consistent with the SEQ ID NO:1 processing group.

[0115] Immunoblotting results confirmed that SEQ ID NO:1 and its homologs can induce phosphorylation of MPK3 and MPK6 in plant cells, initiating intracellular immune signal transduction.

[0116] This embodiment utilizes real-time quantitative PCR (RT-qPCR) technology to detect the transcriptional expression levels of defense-related genes in plants after treatment with SEQ ID NO:1 and its homologous variants. The detection targets are salicylic acid signaling pathway marker gene (PR1), transcription factor gene (WRKY), and respiratory burst oxidase homolog gene (RBOHB).

[0117] In addition, the test results for citrus leaves (such as...) Figure 2As shown in the bottom row, treatment with SEQ ID NO:1 can also induce specific MPK3 and MPK6 phosphorylation bands in citrus leaves, and the signal intensity is significantly enhanced within 10 to 30 minutes after treatment. This confirms that the polypeptide has the same immune-activating activity on citrus crops, providing direct evidence for its application in the control of citrus Huanglongbing.

[0118] S510: Plant Induction Treatment and Sample Collection Four-week-old tobacco plants and one-year-old sweet orange plants were randomly divided into treatment and control groups. The polypeptide of SEQ ID NO:1 was dissolved in sterile deionized water, and Silwet L-77 surfactant was added to a final concentration of 0.02% (v / L) to prepare a solution with a concentration of 1 μmol / L. Apply the treatment solution evenly to both sides of the Tobacco Bunge leaves using a handheld sprayer. The amount of spray should be such that the leaf surface is covered with fine droplets without dripping.

[0119] The control group was sprayed with sterile deionized water containing 0.02% (v / v) Silwet L-77. Following the above method, treatment groups with SEQ ID NO:2 (R13K variant) and SEQ ID NO:3 (V1I variant) were set up, with a peptide concentration of 1 μmol / L in both groups. ).

[0120] Leaf samples were collected at 3, 6, 12, and 24 hours after treatment. Each sampling consisted of three leaves from three different plants, forming a single biological replicate, with three replicates per group. The collected leaves were flash-frozen in liquid nitrogen and then stored at -80°C.

[0121] S520: Total RNA Extraction and cDNA Synthesis Take approximately 100 mg of frozen leaf tissue and grind it into powder in liquid nitrogen. Extract total RNA using a plant total RNA extraction kit. During the extraction process, perform DNase I on-column digestion for 15 minutes to remove residual genomic DNA. Detect RNA concentration using a UV spectrophotometer. The A260 / A280 ratio should be between 1.9 and 2.1.

[0122] One microgram of total RNA was used as a template to synthesize the first strand of cDNA using a reverse transcription kit. The reaction system included Oligo(dT)18 primers, and the reaction program was set as follows: incubation at 42°C for 60 minutes, followed by incubation at 70°C for 5 minutes. The reaction product was diluted 10-fold and used as a qPCR template.

[0123] S530: Real-time quantitative PCR detection Based on the genome sequence of Nicotiana bungeana, the following specific primers were designed: (1) NbFRK1 (pathology-associated protein 1 gene): Upstream primer (F): 5'-GTG GAT ACG GAC AAA ACG-3' Downstream primer (R): 5'-CCT AGC ACA TCC AAC ACG-3' (2) NbWRKY22 (WRKY transcription factor gene): Upstream primer (F): 5'-TCG GAT GCT GCT GAG AGT-3' Downstream primer (R): 5'-CTT GTC CAC GAG GAT GCT-3' (3) NbRBOHB (respiratory burst oxidase homolog B): Upstream primer (F): 5'-ATG GCT GGT GAT GGT TTG-3' Downstream primer (R): 5'-GCA ATC GAA GCT GCT GTA-3' (4) NbActin (internal reference gene): Upstream primer (F): 5'-ATG GCA GAC GGT GAG GAT-3' Downstream primer (R): 5'-GAG GAA GCA AGG ATA GAC-3' Meanwhile, specific primers targeting the sweet orange (Cs) homologous genes CsFRK1, CsWRKY22 and CsRbohB were designed for the detection of citrus samples.

[0124] The reaction was performed on a real-time quantitative PCR instrument. The 20 μL reaction mixture contained: 10 μL SYBR Green Premix, 0.4 μL upstream primer (10 μL...). ), 0.4 μL downstream primer (10 ), 2 μL cDNA template, 7.2 μL nuclease-free water.

[0125] The amplification program was as follows: pre-denaturation at 95°C for 30 seconds; followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. After the cycles, melting curve analysis was performed at a temperature range of 65°C to 95°C at a heating rate of 0.5°C / second, confirming that the product was a single amplification peak.

[0126] S540: Data Computation and Analysis Record the cycle threshold (Ct value) for each sample and use the comparison Ct method ( (Method) to calculate the relative expression level of genes.

[0127] The calculation formula is as follows: Relative expression level =

[0128] =Target gene Ct value - Internal reference gene Ct value; =Processing Group Value - Control Group value; S550: Test Results The test data shows that: NbFRK1 and NbWRKY22 genes: In the SEQ ID NO:1 treatment group, the expression level of these genes showed an increasing trend over time. 24 hours after treatment, the relative expression level of NbFRK1 was 15.6 times that of the control group.

[0129] NbWRKY22 gene: 3 hours after treatment, the relative expression level of NbWRKY22 in the SEQ ID NO:1 treatment group reached 8.4 times that of the control group, and then the expression level decreased, which is consistent with the expression characteristics of an early response gene.

[0130] NbRBOHB gene: 6 hours after treatment, the relative expression level of NbRBOHB in the SEQ ID NO:1 treatment group was 6.2 times that of the control group.

[0131] Homologous variant effects: Both SEQ ID NO:2 and SEQ ID NO:3 treatments induced upregulation of the above genes. At 24 hours post-treatment, SEQ ID NO:2 induced a 12.3-fold relative expression level of NbFRK1 compared to the control group, while SEQ ID NO:3 induced a 14.8-fold relative expression level of NbFRK1 compared to the control group.

[0132] The results confirmed that treatment with SEQ ID NO:1 and its homologous variants could induce upregulation of the transcriptional level of defense-related genes in tobacco leaves.

[0133] This embodiment uses *Pseudomonas syringae* (… P. syringae pv. tomato DC3000 was used as the test pathogen, with *Tobacco Bunsenii* (pv. tomato DC3000) as the test pathogen. N. benthamiana Using SEQ ID NO:1 and its homologs as host plants, the ability of SEQ ID NO:1 and its homologs to inhibit the proliferation of pathogens in plant tissues was evaluated through in vivo inoculation experiments.

[0134] S610: Strain activation and inoculum preparation The strain used in this embodiment is an engineered strain carrying the luciferase reporter gene. *Pseudomonas syringae* DC3000 preserved in glycerol tubes was streaked and inoculated into a solution containing 50 μg / L (…). Incubate King's B solid agar plates containing rifampicin (g / L) at 28°C with inverted incubation for 48 hours. Pick single colonies and transfer them to liquid King's B agar, incubating at 28°C with shaking at 200 rpm until the logarithmic growth phase. Collect bacterial cells by centrifugation using sterile 10 mmol / L (g / L) rifampicin. Magnesium chloride ( Resuspend the bacterial cells in the solution and wash twice. Adjust the bacterial concentration to... (The corresponding live bacteria concentration is approximately) ), used as an inoculum for virus challenge.

[0135] S620: Peptide pretreatment Five-week-old Nicotiana bulbifera plants were randomly divided into five groups. The following treatment solutions were prepared: SEQ ID NO:1 treatment group: SEQ ID NO:1 aqueous solution with a concentration of 1 μmol / L; SEQ ID NO:2 variant treatment group: SEQ ID NO:2 aqueous solution at a concentration of 1 μmol / L; SEQ ID NO:3 variant treatment group: SEQ ID NO:3 aqueous solution at a concentration of 1 μmol / L; Positive control group: 1 μmol / L flg22 aqueous solution; Negative control group: sterile deionized water.

[0136] Using a 1 ml needleless syringe, the treatment solution was injected into the mesophyll tissue from the back of the leaf. The 3rd, 4th, and 5th true leaves of each plant were selected for injection, and the edges of the injection areas were marked. The treated plants were then placed in a light incubator for normal cultivation.

[0137] S630: Pathogen inoculation Inoculation was performed 24 hours after peptide pretreatment. The pathogen inoculum prepared in S610 was aspirated and injected into the same leaf area marked in S620 using a needleless syringe. After inoculation, the plants were kept moist in an environment with a relative humidity of over 90% for 24 hours, and then cultured at normal humidity (approximately 60%).

[0138] Furthermore, in vivo fluorescence imaging monitoring of pathogen proliferation was conducted 3 days post-inoculation. A plant in vivo imaging system was used to detect bioluminescent signals on leaves to visually monitor pathogen proliferation. Leaves were sprayed with a 1 mmol / L solution of D-Luciferin potassium salt, and images were taken after a 5-minute reaction in the dark. Imaging results (e.g.) Figure 4As shown in A and 4C): The inoculated area of ​​the negative control group (CK / EV) showed a strong fluorescent signal (red / warm tone), indicating that the pathogen had multiplied extensively; while the fluorescence signal of the SEQ ID NO:1 treatment group (marked as Palpep / Pal19 in the figure) was weak (blue / cool tone). In addition, as the concentration of the peptide treatment increased (from 100 nM to 1 mM), the fluorescence signal intensity on the leaves gradually decreased, showing a clear dose-dependent effect.

[0139] S640: Record of Onset Symptoms Leaf phenotype was observed 4 days after inoculation.

[0140] The inoculated areas of the leaves in the negative control group showed obvious chlorosis and yellowing, accompanied by water-soaked necrosis, with yellow halos around the lesions.

[0141] In the SEQ ID NO:1 treatment group, the inoculated area of ​​the leaves showed only slight chlorosis, and no necrotic spots were observed or the area of ​​necrotic spots was significantly smaller than that of the negative control group.

[0142] The leaf symptoms in the SEQ ID NO:2 and SEQ ID NO:3 treatment groups were similar to those in the SEQ ID NO:1 treatment group, but the degree of lesions was milder than that in the negative control group.

[0143] S650: Determination of pathogen count in leaves The bacterial growth in leaves was determined using the plate colony counting method.

[0144] S651: Sampling. Four days after inoculation, leaf discs were collected from the center of the inoculation area using a 1 cm diameter punch. Three biological replicates were selected from each group, with each replicate containing two leaf discs.

[0145] S652: Grinding and dilution. Place two leaf discs into a grinding tube containing 1 mL of sterile 10 mmol / L magnesium chloride solution and grind thoroughly into a homogenate. Perform 10-fold serial dilutions on the homogenate to prepare... , , Diluted bacterial solution.

[0146] S653: Spread culture. Take 20 μL of bacterial suspension of each gradient and drop it onto King's B solid plate containing 50 mg / L rifampin. After drying, incubate at 28 degrees Celsius for 36 hours.

[0147] S654: Counting, counting the number of single colonies on the plate, and converting the number of colonies per square centimeter of leaf area based on the dilution factor and sampling area.

[0148] S660: Statistics on Prevention and Control Effectiveness The inhibition rate of each treatment group relative to the negative control group was calculated using the following formula: Antibacterial inhibition rate = (Average bacterial count in negative control group - Average bacterial count in treatment group) / Average bacterial count in negative control group × 100% The statistical results are as follows: Negative control group: The average number of pathogens in the leaves was .

[0149] SEQ ID NO:1 treatment group: The average number of pathogens in the leaves was .

[0150] Calculate the antibacterial rate: .

[0151] The results confirmed that treatment with SEQ ID NO:1 reduced the number of pathogens in the leaves by approximately 90%.

[0152] Homologous variants: The inhibition rate of the SEQ ID NO:2 treatment group was 88.5%, and the inhibition rate of the SEQ ID NO:3 treatment group was 91.2%.

[0153] Analysis of significant differences: Student's t-test was used for statistical analysis. The difference between the SEQ ID NO:1 and variant treatment groups and the negative control group was extremely significant (P<0.01).

[0154] Experimental results confirmed that treatment with SEQ ID NO:1 and its homologs could significantly inhibit the proliferation of Pseudomonas syringae in plant tissues.

[0155] The experimental results showed that although Pseudomonas syringae (DC3000) carries a sequence homologous to SEQ ID NO:1 (VATGNDEQSWAQNRRVELRK), plants still cannot effectively resist its infection (see negative control group).

[0156] Comparison of the homologous sequences of SEQ ID NO:1 and DC3000 reveals that SEQ ID NO:1 possesses a specific amino acid combination (particularly the lysine K at position 12 and a specific conformation at the N-terminus). This experiment confirms that exogenous application of highly active SEQ ID NO:1 (or its potent homologs SEQ ID NO:2 / 3) can "remedy" the plant's deficiency in pathogen recognition and forcibly activate the immune system evaded by pathogens. This demonstrates that the polypeptide described in this invention is not a simple fragment of pathogens, but a highly efficient immune inducer with specific structural advantages that can overcome pathogen immune escape.

[0157] This embodiment describes the formulation, processing technology, and specific field application procedures of an agricultural composition comprising SEQ ID NO:1 or its homologous variants.

[0158] S710: Preparation process of aqueous solution (SL) The aqueous solution is suitable for foliar spraying and trunk injection. Formulation composition (taking the preparation of 1 liter of formulation as an example): Active ingredient: SEQ ID NO:1 peptide, added in amounts of 1 to 10 grams (corresponding to a content of 0.1% to 1.0%). Wetting agent: Polyoxyethylene dehydrated sorbitan monolaurate (Tween-20), added in amounts of 10 to 50 grams; Stabilizer: Disodium ethylenediaminetetraacetate (EDTA-2Na), 1 gram added; Preservative: Sodium benzoate, 2 grams added; Solvent: Deionized water, add to a total volume of 1000 ml.

[0159] Processing steps: Add 80% of the formulated amount of deionized water to a reactor equipped with a mechanical stirrer. Start the stirrer and set the speed to 200 to 300 rpm. Add disodium ethylenediaminetetraacetate and sodium benzoate sequentially, stirring for 5 to 10 minutes until the solids are completely dissolved. Slowly add Tween-20 dropwise, continuing to stir for 15 minutes. Finally, add accurately weighed SEQ ID NO:1 peptide lyophilized powder, and maintain low-speed stirring at room temperature for 30 minutes until the solution is clear and transparent. Filter the resulting solution through a 0.45-micron pore size filter membrane and bottle to obtain the aqueous product.

[0160] S720: Preparation process of wettable powder (WP) This wettable powder is suitable for large-area spraying or root application. Formulation composition (by weight percentage): Active ingredient: SEQ ID NO:1 polypeptide (or SEQ ID NO:2 variant), in a concentration of 1.0% to 5.0%; Dispersant: Sodium lignosulfonate, content of 5.0% to 10.0%; Wetting agent: Sodium dodecyl sulfate (SDS), content of 2.0% to 4.0%; Carrier: Kaolin (or diatomaceous earth), make up to 100%.

[0161] Processing steps: The polypeptides, sodium lignosulfonate, sodium dodecyl sulfate, and kaolin are mixed and coarsely crushed in a specific ratio. The coarse mixture is then fed into an air jet mill for ultrafine grinding under compressed air pressure of 0.6 to 0.8 MPa. The fineness of the grinding is controlled so that more than 98% of the particles have a diameter less than 45 micrometers (325 mesh). The ground material is then mixed again in a mixer until homogeneous, and samples are taken for testing. When the suspension rate is greater than 70% and the wetting time is less than 2 minutes, it is considered qualified and quantitatively packaged.

[0162] S730: Preparation process of sustained-release granules (GR) Granules are applied to the soil and absorbed through the roots. Formulation composition (by weight percentage): Active ingredient: SEQ ID NO:1 peptide, in a concentration of 0.5% to 2.0%; Binder: Sodium carboxymethyl cellulose (CMC-Na), with a content of 1.0% to 3.0%; Disintegrant: Bentonite, content 10.0%; Carrier: Fine sand, replenished to 100%.

[0163] Processing steps: Mix the peptides, bentonite, and fine sand evenly. Dissolve sodium carboxymethyl cellulose in water to prepare a 2% binder solution. Place the mixed powder in a disc granulator and spray in the binder solution for rolling granulation. Control the particle diameter between 0.5 and 1.5 mm. Dry the obtained wet granules in a 50°C forced-air drying oven until the moisture content is below 5%. Sieve to remove powder and large pieces to obtain the slow-release granules.

[0164] S740: Field Application Methods Choose the following application method based on the crop type and the location of the disease: 1. Foliar spraying For bacterial leaf spot disease of tobacco, vegetables, and fruit trees, the procedure is as follows: Dilute the aqueous solution prepared by S710 or the wettable powder prepared by S720 with water to adjust the final peptide concentration to 1-10 μM. Use a mist sprayer to spray both sides of the plant leaves, spraying only until the leaves are moist. Spray once every 7 to 10 days during the disease-prone period, for 2 to 3 consecutive applications.

[0165] 2. Root irrigation and application For crops with soil-borne diseases or those requiring long-term protection, the procedure is as follows: Apply the granules prepared by S730 to the planting hole when sowing or transplanting the crop, at a rate of 1 to 5 grams per plant, followed by covering with soil and watering. Alternatively, dilute the wettable powder prepared by S720 by 500 to 1000 times and irrigate directly into the root soil.

[0166] 3. Trunk injection (for citrus Huanglongbing) To address the colonization of Huanglongbing (HLB) in citrus by the phloem, an injection method is used for application. The procedure is as follows: Use an aqueous solution prepared with S710, applying the undiluted solution directly or diluted 2 to 5 times. Drill a hole at a 45-degree angle on the trunk of the citrus tree, 20 to 30 cm above the ground. The hole should penetrate the phloem and reach approximately 1 to 2 cm into the xylem, with a diameter of 4 to 6 mm. Attach a trunk injector or hanging bag and inject 10 to 50 ml of the solution (adjusting according to tree age). The solution is transported upwards via transpiration from the xylem and laterally to the phloem, inducing a defensive response in the phloem cells to inhibit HLB.

[0167] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A plant immune-inducing polypeptide, characterized in that, The amino acid sequence of the plant immune-inducing polypeptide is selected from any of the following: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) The amino acid sequence as shown in SEQ ID NO:2; (3) The amino acid sequence as shown in SEQ ID NO:3; (4) An amino acid sequence that, based on the sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, has one to three amino acid residues that have been conservatively substituted, deleted or added, and that can induce plants to produce reactive oxygen species or activate the mitogen-activated protein kinase pathway. (5) An amino acid sequence that is more than 85% identical to the sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and can induce plants to produce reactive oxygen species or activate the mitogen-activated protein kinase pathway.

2. A polynucleotide encoding the plant immune-inducing polypeptide of claim 1, characterized in that, The nucleotide sequence of the polynucleotide is shown in SEQ ID NO:4, or it is a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:

3.

3. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 2; wherein the recombinant vector is a plant expression vector, and the polynucleotide is inserted between the CaMV 35S promoter and the NOS terminator of the recombinant vector.

4. An engineered bacterial strain, characterized in that, The engineered strain comprises the recombinant vector of claim 3; wherein the engineered strain is Agrobacterium tumefaciens (…). Agrobacterium tumefaciens ).

5. An agricultural composition, characterized in that, The agricultural composition comprises an effective amount of the plant immune-inducing polypeptide of claim 1 as an active ingredient, and a pesticide-acceptable carrier or adjuvant; the formulation of the agricultural composition includes aqueous solutions, wettable powders, or sustained-release granules.

6. The agricultural composition according to claim 5, characterized in that: The aqueous formulation comprises the plant immune-inducing polypeptide, polyoxyethylene sorbitan monolaurate as a wetting agent, disodium ethylenediaminetetraacetate as a stabilizer, sodium benzoate as a preservative, and deionized water; the wettable powder comprises the plant immune-inducing polypeptide, sodium lignosulfonate as a dispersant, sodium dodecyl sulfate as a wetting agent, and kaolin or diatomaceous earth as a carrier; the slow-release granules comprise the plant immune-inducing polypeptide, sodium carboxymethyl cellulose as a binder, bentonite as a disintegrant, and fine sand as a carrier.

7. The use of the plant immune-inducing polypeptide of claim 1, the polynucleotide of claim 2, the engineered strain of claim 4, or the agricultural composition of claim 5 in the preparation of formulations for the prevention and control of bacterial plant diseases.

8. The application according to claim 7, characterized in that, The aforementioned bacterial plant diseases include: spp. of phloem ( Candidatus Liberibacter )include Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, Candidatus Liberibacter Americanus Citrus Huanglongbing caused by, or by ‌Candidatus Liberibacter solanacearum Diseases caused by; Pseudomonas spp. ( Pseudomonas ), including Pseudomonas syringae, which causes leaf spot or canker. Xanthomonas ( ) Xanthomonas ), including those that cause citrus canker. Xanthomonas citrisubsp. citri .

9. A method for controlling bacterial diseases of plants, characterized in that, This includes applying the agricultural composition of claim 5 to plants; the method of application is selected from foliar spraying, root irrigation, root application, or trunk injection.

10. The method according to claim 9, characterized in that: In the foliar spraying, the agricultural composition is diluted to a final concentration of 1 to 10 micromoles per liter for the plant immune-inducing polypeptide; in the trunk injection, a hole is drilled at a 45-degree angle at a distance of 20 to 30 centimeters from the ground on the plant trunk using an electric drill, the hole penetrating the phloem and entering the xylem to a depth of 1 to 2 centimeters, and then the aqueous form of the agricultural composition is injected.

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