The genes and primers used in this experiment were synthesized by Tsingke Biotechnology, and the PCR reagents were purchased from YEASON Biotechnology. The primers and plasmids are listed in Table 1.

2.1 Plasmid Verification
PCR verification was performed on the genes synthesized by the company using primers pET-28a-F and pET-28a-R. The PCR system is shown in Table 2. The program was set as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 58°C for 15 s, and extension at 72°C for 45 s, with a final extension at 72°C for 4 min.

After PCR, gel electrophoresis was conducted following the steps below:
① Measure 50 mL of 1×TAE electrophoresis buffer (Preparation of 50×TAE stock solution: Mix 242 g of Tris, 37.2 g of disodium ethylenediaminetetraacetate dihydrate, and 57.1 mL of glacial acetic acid, then add 1 L of deionized water, stir and mix well; dilute 50-fold with deionized water before use). Add 0.5 g of agarose for electrophoresis, mix well, and heat in a microwave oven at medium-high power for 2-3 min to fully dissolve the agarose. Keep an eye on it to prevent the boiling liquid from overflowing. After cooling slightly, add 5 μL of DNA safe dye and mix well.
② Take the organic glass inner tank, clean and dry it; place the comb and adjust the distance between the bottom edge of the comb and the electrophoresis plate, which is generally 1-2 mm. Place the organic glass inner tank on a horizontal mold.
③ Pour the agarose solution into the electrophoresis plate, let it stand, and do not move it.
④ After the gel is completely solidified, gently take out the comb and place the gel into the electrophoresis tank.
⑤ Add 1×TAE electrophoresis buffer into the electrophoresis tank so that the electrophoresis buffer covers the surface of the agarose gel by approximately 1-2 mm, and perform electrophoresis at 150 V for 30 min.

(1) Inoculate the primary seed culture and incubate overnight at 37°C with shaking at 200 rpm.
(2) Inoculate 1% of the primary seed culture into 500 mL of secondary seed medium, and incubate at 37°C with shaking at 160 rpm until the OD₆₀₀ value reaches 0.6-0.8. (Under the condition of 1‰ antibiotic, this process usually takes 2 hours.)
(3) Place the shake flask at the induction temperature of 18°C. After cooling down, add 200 μL of 1 M IPTG, and incubate at 18°C with shaking at 160 rpm for 14-18 hours to harvest the bacteria. (Tips: The induction temperature and IPTG concentration need to be optimized according to the specific protein.)
(4) Harvest the bacteria using a bacteria collection bottle, and centrifuge at 4000 rpm for 20 min in a low-temperature centrifuge. (Prepare the relevant reagents before harvesting the bacteria.)
(5) Discard the supernatant, resuspend the pellet with 30 mL of Buffer A (20 mM Tris-HCl, 500 mM NaCl, pH 8.0; the pH is determined by the isoelectric point of the protein; filter through a 0.22 μm or 0.45 μm filter membrane), transfer to a 50 mL EP tube, and centrifuge at 8000 rpm for 5 min in a low-temperature centrifuge to completely remove the LB medium.
(6) Resuspend the pellet obtained by centrifugation with 30-40 mL of Buffer A, and prepare for bacterial cell disruption using a high-pressure cell disruptor. From this point on, all operations involving the bacterial cells should be carried out in a low-temperature refrigerator or on ice to prevent protein precipitation.
(7) The procedure for disrupting bacterial cells with a high-pressure cell disruptor is as follows: Turn on the machine in advance to cool it down to 4°C, clean it with pure water, rinse it with Buffer A, increase the pressure to 850 bar-950 bar, load the sample for disruption for 2 min, reduce the pressure for 1 min, and repeat this process 1-2 times (or disrupt directly without reducing the pressure for 5 min; note that the machine should not overheat) until the outflow of the disrupted bacterial suspension is no longer viscous and the bacterial suspension becomes clear. After the disruption is completed, clean the machine with Buffer A, then with pure water, and finally clean and seal it with 20% ethanol, then turn off the machine.
(8) Transfer the disrupted bacterial suspension to an ultracentrifuge tube, and centrifuge at 10000 rpm at 4°C for at least 40 min.
(9) After centrifugation, use a syringe to aspirate the supernatant, filter it through a 0.22 μm or 0.45 μm needle filter, collect the filtrate into a new 50 mL EP tube, and then load the filtrate onto a nickel column as soon as possible to obtain the target protein.

(1) Preparation of the nickel column: If the nickel column needs to be thoroughly cleaned, 100 mM EDTA and 100 mM NaOH should be used to remove impurities, and then 100 mM nickel sulfate should be reloaded to obtain a regenerated new nickel column. (Rinse with pure water between the alternation of different solutions for transition.)
(2) Take out the nickel column (usually with a column volume of 5 mL) at 4°C, rinse it with pure water, equilibrate it with Buffer A for loading (approximately 30 mL), load the protein sample 1-2 times, remove impurities with a high-salt solution (20 mM Tris-HCl, 2 M NaCl, pH 8.0; filter through a 0.22 μm or 0.45 μm filter membrane) (approximately 30 mL), and equilibrate with Buffer A (approximately 30 mL). (Tips: The flow rate should not be too fast during all loading processes; for machines with flow rate control, the flow rate should be set to 5-10 mL/min.)
(3) Wash away impurities with 80 mM imidazole (prepared by mixing Buffer A and Buffer B (20 mM Tris-HCl, 500 mM NaCl, 500 mM Imidazole, pH 8.0; the pH is determined by the isoelectric point of the protein; filter through a 0.22 μm or 0.45 μm filter membrane)). The appropriate imidazole concentration for impurity washing needs to be optimized in the early stage of the experiment to remove as many impurity proteins as possible while retaining the target protein. The volume of the washing solution is approximately 30-50 mL.
(4) Elute the target protein with a high-concentration imidazole solution (generally 300 mM-500 mM imidazole, prepared by mixing Buffer A and Buffer B). During elution, Coomassie Brilliant Blue reagent can be used to detect whether protein is flowing out. After protein outflow is detected, collect approximately 3-10 mL of the eluate.
(5) Cleaning and preservation of the nickel column: Wash the nickel column with Buffer B until no protein is detected in the eluate by Coomassie Brilliant Blue reagent (generally 50-100 mL of Buffer B is used). After rinsing with pure water, store the nickel column at 4°C. It can be used directly for the next purification of the same protein.

(1) Evaluation of protein concentration: NanoDrop is generally used to detect UV₂₈₀, but its accuracy is not high.
(2) Evaluation of protein purity: Perform SDS-PAGE gel electrophoresis. The samples to be loaded are in the following order: ① supernatant after bacterial cell disruption, ② pellet after bacterial cell disruption, ③ flow-through from the nickel column during loading, ④ impurity proteins flowing out during impurity washing, ⑤ eluate containing the target protein. Generally, the protein concentration of the samples is relatively high, so dilution is required. Set the electrophoresis conditions to 150 V for 40 min.
(3) Concentration of protein samples: Use an ultrafiltration tube with an appropriate pore size, centrifuge at 4000 rpm, and detect the protein sample concentration every 10 min until the experimental requirements are met.

Tomato plants: Select tomato seedlings at the 4-5 leaf stage with consistent growth status.
Ralstonia solanacearum strains: Use a standard strain of Ralstonia solanacearum (e.g., Ralstonia solanacearum GMI1000). Inoculate it on NA medium (Nutrient Agar), incubate at 28°C for 48 hours, and dilute with sterile water to OD₆₀₀=0.5 (approximately 10⁸ CFU/mL) to prepare a bacterial suspension for later use.
Functional bacterial solution: Culture in LB medium at 37°C for 8 hours, and dilute with sterile water to OD₆₀₀=0.5 to prepare a bacterial suspension for later use.
Reagents and equipment: NA medium, sterile water, pipettes, culture pots (10 cm in diameter), greenhouse temperature control equipment (25-30°C, 70% humidity), colony counting plates, PCR instrument (for pathogen quantification), etc.

Randomly divide the tomato seedlings into 4 groups, with 6 plants in each group and 3 replicates set. The specific grouping is as follows:
•Blank control group: Only water with sterile water, no Ralstonia solanacearum infection treatment.
•Ralstonia solanacearum control group: Water with Ralstonia solanacearum suspension (10 mL per plant), no bacterial solution application.
•Negative control group (non-functional Escherichia coli bacterial solution group): First water with negative control Escherichia coli bacterial solution (10 mL per plant), and water with Ralstonia solanacearum suspension (10 mL per plant) 2 hours later.
•Treatment group (functional Escherichia coli bacterial solution group): First water with functional Escherichia coli bacterial solution (10 mL per plant), and water with Ralstonia solanacearum suspension (10 mL per plant) 2 hours later.

Pretreatment and infection: Water each group according to the above grouping design, and inject slowly along the roots to avoid liquid overflow. After treatment, place all plants in the same greenhouse environment, and record the growth status daily (16 h light/8 h dark, temperature 28°C).
Disease investigation cycle: Starting from the 3rd day after infection, observe and record the disease occurrence of tomato plants daily. Refer to the Ralstonia solanacearum disease grading standard (Grade 0: No symptoms; Grade 1: 1-2 leaves wilted; Grade 2: 3-5 leaves wilted; Grade 3: Whole plant wilted; Grade 4: Plant dead) to count the disease grades and calculate the disease index (Disease index = Σ (Disease grade × Number of plants) / (Highest grade × Total number of plants) × 100).
Determination of erucamide content by liquid chromatography: On the 21st day, randomly select 3 plants from each group, and collect the substrate leachate around the roots and root tissues (0.5 g) respectively. For the substrate leachate, directly filter it through a 0.22 μm filter membrane and take 1 mL of the filtrate for testing. For the root tissues, add 2 mL of methanol for grinding and homogenization, perform ultrasonic extraction at 4°C for 30 minutes (power 200 W, 3 seconds of ultrasound and 3 seconds of pause), centrifuge at 12000 rpm for 10 minutes, take the supernatant, and re-extract the residue with 1 mL of methanol once. Combine the two supernatants, filter through a 0.22 μm filter membrane, and store the filtrate at 4°C for testing.
Chromatographic conditions: A C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was used; the mobile phase was methanol-0.05 mol/L ammonium acetate solution (85:15, v/v); the flow rate was 1.0 mL/min; the column temperature was 30°C; the detection wavelength was 205 nm; the injection volume was 20 μL.
Standard curve drawing: Accurately weigh the erucamide standard, dissolve it in methanol and dilute it into standard solutions with concentrations of 1 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 50 μg/mL, and 100 μg/mL. Inject and determine according to the above chromatographic conditions, with the peak area as the ordinate and the erucamide concentration as the abscissa, draw the standard curve.
Sample determination: Inject and determine the processed samples to be tested according to the above chromatographic conditions, and calculate the erucamide content in the samples (μg/mL or μg/g) according to the standard curve.

Disease dynamic curve: With the number of days after infection as the horizontal axis and the disease index as the vertical axis, draw the disease trend curve of each group, and compare the differences in disease progression between the treatment group and the control group.
Plant survival rate: Count the number of surviving plants in each group on the 21st day after infection, and calculate the survival rate (Survival rate = Number of surviving plants / Total number of plants × 100%).
Comparative analysis of erucamide content: Taking the 21st day after infection as the time point and the erucamide content as the vertical axis, draw the final erucamide content of each group, and analyze the comparison of erucamide content among different experimental groups. At the same time, conduct a correlation analysis between the erucamide content and the disease index in the treatment group to explore the relationship between erucamide content and disease resistance effect.

Mix our protein-expressing bacterial strain, sucrose, and 1% sodium alginate solution, and drop the mixture into a Ca²⁺ solution. Sodium alginate reacts with calcium ions to form a water-insoluble calcium alginate shell on the outside, while encapsulating the engineered bacteria and sucrose to form smaller beads inside the calcium alginate. Then, encapsulate them in a PVA film of a certain size, and finally seal with a heat sealer.