A ralstonia solanacearum raa r protein inhibitor and application thereof

Abstract

The application relates to a Ralstonia solanacearum RaaR protein inhibitor and application thereof, and belongs to the technical field of protein inhibitors. The Ralstonia solanacearum RaaR protein inhibitor of the application is a 2-amino-4-fluorobenzoic acid (2A4FBA) compound. The application is screened from 9 kinds of anthranilic acid analogs based on the influence on transcriptional expression of Ralstonia solanacearum quorum sensing synthesis genes, and finally determines that 2-amino-4-fluorobenzoic acid is a target RaaR protein inhibitor. At a concentration of 10 muM, the compound has no significant influence on the growth state of Ralstonia solanacearum, but can effectively inhibit the transcriptional expression of quorum sensing synthesis genes. When the concentration of 2-amino-4-fluorobenzoic acid is 10 muM, and the titer of Ralstonia solanacearum bacteriophage PN12-4 is 10 4 PFU / mL, the mortality of tomato plants can be reduced.

Description

Technical Field

[0001] This application relates to the field of protein inhibitor technology, and in particular to a RaaR protein inhibitor of Ralstonia solanacearum and its application. Background Technology

[0002] Ralstonia solanacearum, also known as the bacterial wilt bacteria ( Ralstonia solanacearum Ralstonia solanacearum is a Gram-negative, aerobic, soil-borne pathogenic bacterium. This bacterium can invade plants through the root system, multiply rapidly in the vascular bundles, causing blockages and ultimately leading to the plant's wilting and death due to dehydration. Ralstonia solanacearum is pathogenic to more than 50 families and over 200 genera of plants, including important economic crops such as bananas, peanuts, eggplants, and tomatoes, causing enormous economic losses to global agricultural production.

[0003] Quorum sensing is a communication mechanism in which bacteria coordinate group behavior through small molecule compounds secreted by themselves, mainly regulating biological processes such as biofilm formation, motility, and the production of virulence factors. Currently, four quorum sensing systems have been identified in *Ralstonia solanacearum*: phc system, sol system, trp System and ras The system. Among them, phc The system is the core regulatory system for the pathogenicity of Ralstonia solanacearum; sol The system is an AHL-mediated swarm sensing system; trp The system is a newly discovered swarm sensing system in recent years, based on... trp The EG-encoded anthranilic acid synthase synthesizes the anthranilic acid signaling molecule. After anthranilic acid binds to its receptor protein RaaR, it regulates important biological functions and the expression of virulence factors in *Ralstonia solanacearum*. Furthermore, *Ralstonia solanacearum* also contains... ras This system plays an important role in the expression regulation of Ralstonia solanacearum virulence factors and in the process of host infection.

[0004] Currently, the main methods for controlling bacterial wilt include breeding resistant varieties, chemical control, and biological control. Among these, quorum sensing inhibitors are a novel class of control agents that weaken the pathogenicity of bacteria by interfering with or blocking the bacterial quorum sensing system. Unlike traditional fungicides, quorum sensing inhibitors do not directly kill bacteria, but rather inhibit pathogenic behaviors such as the expression of virulence factors and biofilm formation by blocking signal communication between bacteria. This reduces the pathogenicity of the pathogen without causing selective pressure, effectively reducing plant mortality. Therefore, quorum sensing inhibitors are considered an eco-friendly, green control strategy that is less likely to induce drug resistance. 2-Amino-4-fluorobenzoic acid (2A4FBA) is an o-aminobenzoic acid analog that can inhibit the activity of *Ralstonia solanacearum*. trpThis compound targets the quorum sensing system, weakening its virulence. Compared to traditional quorum sensing inhibitors, this compound has a more clearly defined target, acting on *Ralstonia solanacearum*. trp The RaaR protein in the quorum sensing system exerts an inhibitory effect. On the other hand, Ralstonia solanacearum phage (… Ralstonia solanacearum phage With its high host specificity and strong bacterial lysis ability, 2-amino-4-fluorobenzoic acid is also a novel green biological control method. When 2-amino-4-fluorobenzoic acid is applied in combination with bacteriophage PN12-4, it inhibits the quorum sensing system of *Ralstonia solanacearum* and reduces its pathogenicity. Simultaneously, the bacteriophage specifically recognizes and lyses *Ralstonia solanacearum* host cells, directly reducing the pathogen population. The synergistic effect of these two mechanisms weakens the pathogenicity of *Ralstonia solanacearum* while effectively reducing its population density, thus achieving a control effect superior to that of a single agent. Summary of the Invention

[0005] In view of this, this application provides a RaaR protein inhibitor of Ralstonia solanacearum and its application. The RaaR protein inhibitor of Ralstonia solanacearum has an inhibitory effect on the quorum sensing system of Ralstonia solanacearum and has a good effect on preventing bacterial wilt caused by Ralstonia solanacearum. It has good potential application value in the prevention and control of bacterial wilt and can effectively overcome the defects of the above-mentioned prior art.

[0006] The first aspect of this application provides a RaaR protein inhibitor of Ralstonia solanacearum, wherein the RaaR protein inhibitor of Ralstonia solanacearum is a 2-amino-4-fluorobenzoic acid compound.

[0007] This application screened nine anthranilic acid analogues based on their effects on the transcriptional expression of quorum sensing synthesis genes in *Ralstonia solanacearum*, ultimately identifying 2-amino-4-fluorobenzoic acid as the target RaaR protein inhibitor. At a concentration of 10 μM, this compound had no significant effect on the growth status of *Ralstonia solanacearum*, but effectively inhibited the transcriptional expression of its quorum sensing synthesis genes. When the concentration of 2-amino-4-fluorobenzoic acid was 10 μM, combined with other compounds, the potency was 10... 4 When the concentration of Ralstonia solanacearum phage PN12-4 PFU / mL was increased, the mortality rate of tomato plants was reduced.

[0008] The RaaR protein inhibitor obtained in this application—2-amino-4-fluorobenzoic acid—exhibits a good inhibitory effect on the quorum sensing system of Ralstonia solanacearum. Its combined application with Ralstonia solanacearum bacteriophage PN12-4 can effectively reduce the mortality rate of tomato plants, thus serving as an effective means of controlling tomato bacterial wilt caused by Ralstonia solanacearum.

[0009] The second aspect of this application also provides the use of the aforementioned Ralstonia solanacearum RaaR protein inhibitor in the preparation of anti-Ralstonia solanacearum drugs.

[0010] A third aspect of this application also provides an anti-bacterial wilt agent, including the aforementioned Ralstonia solanacearum RaaR protein inhibitor.

[0011] Preferably, it also includes Ralstonia solanacearum phage PN12-4.

[0012] Preferably, the concentration of the Ralstonia solanacearum RaaR protein inhibitor is not less than 1 μM.

[0013] Preferably, the concentration of the Ralstonia solanacearum RaaR protein inhibitor is 1 μM, 5 μM or 10 μM.

[0014] Preferably, the titer of the Ralstonia solanacearum phage PN12-4 is not less than 10. 4 PFU / mL.

[0015] Preferably, the titer of the Ralstonia solanacearum phage PN12-4 is 10. 4 PFU / mL.

[0016] Preferably, the antibacterial agent is in the form of an aqueous solution.

[0017] The fourth aspect of this application also provides the application of the aforementioned antibacterial wilt agent in tomato plants.

[0018] Compared with the prior art, this application has the following advantages:

[0019] (1) This application provides a RaaR protein inhibitor—2-amino-4-fluorobenzoic acid. Compared with traditional pesticides for controlling bacterial wilt, this inhibitor acts on the quorum sensing system of Ralstonia solanacearum. By inhibiting the regulatory activity of the RaaR transcript of the anthranilic acid receptor, it affects the expression of Ralstonia solanacearum virulence factors, thereby inhibiting the virulence of Ralstonia solanacearum and reducing the mortality rate of tomato plants. This provides an inhibitor source and control strategy for the targeted quorum sensing control of Ralstonia solanacearum.

[0020] (2) This application also found that 2-amino-4-fluorobenzoic acid can be used in combination with Ralstonia solanacearum phage to enhance the infection and lysis of Ralstonia solanacearum by Ralstonia solanacearum phage. The number of Ralstonia solanacearum colonized in the roots and stems of tomatoes was significantly reduced compared with the use of inhibitor alone, and the survival rate of tomato seedlings was also improved. The control effect reached 1+1 greater than 2, providing a new strategy for the subsequent combined use of Ralstonia solanacearum and Ralstonia solanacearum phage to control Ralstonia solanacearum disease. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the description of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 A is phcB Screening diagram of anthranilic acid analogs for β-galactosidase activity. Figure 1 B is solI Screening chart of anthranilic acid analogs for β-galactosidase activity. Data in the chart are expressed as mean ± standard deviation (n=3). Statistical analysis was performed. express P <0.05, express P <0.01, express P <0.001;

[0023] Figure 2 The one-step growth curve of 2-amino-4-fluorobenzoic acid;

[0024] Figure 3 This diagram illustrates the control of bacterial wilt in tomatoes by 2-amino-4-fluorobenzoic acid.

[0025] Figure 4 The survival rate of tomato plants treated with 2-amino-4-fluorobenzoic acid alone is shown in the figure.

[0026] Figure 5 Figure A shows the colony count of *Ralstonia solanacearum* strain from tomato roots treated with 2-amino-4-fluorobenzoic acid alone. Figure 5 Figure B shows the colony count of *Ralstonia solanacearum* strains from tomato stems treated with 2-amino-4-fluorobenzoic acid alone. Data in the figure are expressed as mean ± standard deviation (n=3). Statistical analysis is indicated by *. P <0.05, ** indicates P <0.01, *** indicates P <0.001;

[0027] Figure 6 A is a proteoglycan image of RaaR. Figure 6 B represents the ITC detection chromatogram of RaaR and 2-amino-4-fluorobenzoic acid;

[0028] Figure 7 The diagram illustrates the control of bacterial wilt in tomatoes using a combination of 2-amino-4-fluorobenzoic acid and bacteriophage.

[0029] Figure 8A graph showing the survival rate of tomato plants when 2-amino-4-fluorobenzoic acid is used in combination with bacteriophage.

[0030] Figure 9 Figure A shows the colony count of *Ralstonia solanacearum* strains from tomato roots after the combined application of 2-amino-4-fluorobenzoic acid and bacteriophage. Figure 9 Figure B shows the colony count of *Ralstonia solanacearum* strains from tomato stems after the combined application of 2-amino-4-fluorobenzoic acid and bacteriophage. Data in the figure are expressed as mean ± standard deviation (n=3). Statistical analysis is indicated by ***. P <0.001;

[0031] Figure 10 The structural map of the recombinant protein expression vector pMAL-C5X-RaaR DNA. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0033] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0034] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0035] In the following examples, CPG liquid medium consisted of 10 g peptone, 5 g glucose, 1 g acid-hydrolyzed casein, and 1 L deionized water, pH 7-8. CPG solid medium consisted of 5 g glucose, 10 g peptone, 1 g acid-hydrolyzed casein, 3% agar powder, and 1 L deionized water, pH 7-8. Semi-solid medium consisted of 16 g tryptone, 6 g agar, and 1 L deionized water, pH 7-8. LB liquid medium consisted of 10 g peptone, 5 g yeast extract, 10 g NaCl, and 1 L deionized water, pH 7-8.

[0036] Example 1: Determination of transcriptional expression level of Ralstonia solanacearum quorum sensing signal synthase gene

[0037] pME2 is a broad-spectrum host-range reporter plasmid whose backbone carries a promoter-free structure. lacZ Reporter gene. In this embodiment, the plasmid is used as a reporter vector to construct a promoter activity detection system. The system detects promoter activity by measuring the activity of the reporter strain. lacZGene expression can be compared and analyzed to determine the activity of inserted promoters in the report strains. In the β-galactosidase activity assay, using o-nitrobenzene-β-D-galactoside (ONPG) as a substrate, the activity of β-galactosidase can be relatively quantitatively analyzed by observing color changes in the culture medium. phcB and solI Gene promoters and vector pME2- lacZ (Constructed from pME6010 plasmid with the lacZ gene added; pME6010 plasmid catalog number: V004780; NovoPro) lacZ For gene front-end ligation, the ligation system consisted of: 3 µL promoter gene, 1 µL digested vector pME2-lacZ, 5 µL 2×Ezmax Universal CloneMix (catalog number: 24305; TOLOBIO), and 11 µL ddH2O; ligation conditions were 37℃ for 30 min. The ligation product was transformed into DH5α competent cells, and positive clones were screened on LB agar plates (containing tetracycline and X-gal). Blue single clones were picked for colony PCR, and the identified positive clones were sequenced to obtain the recombinant plasmids pME2-pphcB-lacZ and pME2-psolI-lacZ. The constructed pME2-pphcB-lacZ and pME2-psolI-lacZ vectors were introduced into GMI1000 strain via electroporation, followed by screening on CPG solid medium containing tetracycline and X-gal to obtain reporter strains. Single colonies of the reported strain were cultured overnight in CPG liquid medium at 28°C. The overnight culture was then diluted with CPG liquid medium to an OD value of [missing value]. 600After the concentration was set to 0.01, tetracycline was added to the bacterial culture. The mixture was divided into a solvent control group and a compound treatment group. Solvent control group: 10 mL of the above bacterial culture was added + 10 µL DMSO. The compound treatment groups were as follows: N-(3-trifluoromethylphenyl)-o-aminobenzoic acid group: 10 mL of the above bacterial culture + 10 µL of N-(3-trifluoromethylphenyl)-o-aminobenzoic acid DMSO solution; 2-amino-5-hydroxybenzoate group: 10 mL of the above bacterial culture + 10 µL of 2-amino-5-hydroxybenzoate DMSO solution; 2-amino-5-iodobenzoic acid group: 10 mL of the above bacterial culture + 10 µL of 2-amino-5-iodobenzoic acid DMSO solution; 2-amino-4-fluorobenzoic acid group: 10 mL of the above bacterial culture + 10 µL of 2-amino-4-fluorobenzoic acid DMSO solution; 5-bromo-o-aminobenzoate group: 10 mL of the above bacterial culture + 10 µL of 5-bromo-o-aminobenzoate DMSO solution; 2-amino-5-nitrobenzoic acid group: 10 mL of the above bacterial culture + 10 µL of 2-amino-5-hydroxybenzoate DMSO solution. DMSO solution of 2-amino-5-nitrobenzoic acid; methyl 2-amino-3,5-dibromobenzoate group: 10 mL of the above bacterial culture + 10 µL of DMSO solution of methyl 2-amino-3,5-dibromobenzoate; 2-amino-3,5-dibromobenzaldehyde group: 10 mL of the above bacterial culture + 10 µL of DMSO solution of 2-amino-3,5-dibromobenzaldehyde; N-phenyl-o-aminobenzoic acid group: 10 mL of the above bacterial culture + 10 µL of DMSO solution of N-phenyl-o-aminobenzoic acid, with a final concentration of 100 µM for all nine compounds. The bacterial culture was zeroed using sterile CPG liquid medium containing an equal volume of DMSO as a blank, and the OD of the bacterial culture was measured using an ultra-micro spectrophotometer. 600 This overcomes errors caused by DMSO. Cultivate to the OD value of the bacterial culture. 600 =1.0, collect 200 µL of bacterial culture by centrifugation, and resuspend the cells in 600 µL of Z-Buffer (60 mM Na2HPO4, 45 mM NaH2PO4, 9.4 mM KCl, 1 mM MgSO4, 3.5 mM 2-mercaptoethanol). Then, add 60 µL of 0.1% SDS and 60 µL of chloroform sequentially for 5 min each. Quickly add 600 µL of ONPG and incubate at 37°C until the liquid turns yellow (reaction time not exceeding 30 min), recording the incubation time. Immediately stop the reaction by adding 200 µL of 1 M Na2CO3. Centrifuge at 12000 r / min for 5 min, and measure the OD of the supernatant. 420 Values ​​and OD 550 The value is calculated according to the formula. T(min) is the water bath time.

[0038] ;

[0039] Depend on Figure 1 As shown in Figures A and B, the activity of β-galactosidase in *Ralstonia solanacearum* was lowest after the addition of the compound 2-amino-4-fluorobenzoic acid, indicating that 2-amino-4-fluorobenzoic acid has a quorum sensing signal synthase gene effect in *Ralstonia solanacearum*. phcB and solI The inhibition of transcriptional expression was the most significant.

[0040] Example 2: One-step growth curve of 2-amino-4-fluorobenzoic acid

[0041] The overnight culture was diluted with CPG liquid medium to OD. 600 After reaching a concentration of 0.01, the cells were divided into a solvent control group and a compound treatment group. Solvent control group: 10 mL of the above bacterial culture was added + 10 µL of DMSO. Compound treatment group: 10 mL of the above bacterial culture was added + 10 µL of a DMSO solution of 2-amino-4-fluorobenzoic acid. The final concentrations of 2-amino-4-fluorobenzoic acid were 1 µM, 5 µM, and 10 µM, respectively (i.e., corresponding to...). Figure 2 (Four groups in the text). 200 µL of diluted bacterial solution was added to each well of a 96-well plate. Corresponding background correction wells were set up, with the concentration of DMSO or other compounds identical to the corresponding treatment groups, except that no *Ralstonia solanacearum* bacterial solution was added to eliminate background interference from the culture medium. The growth of *Ralstonia solanacearum* was monitored for 72 hours using a Bioscreen-C automated growth curve analyzer at 28°C and in low-speed oscillation mode. The data were then exported and growth curves were plotted. Figure 2 It was found that, compared with the GMI1000+DMSO group, at concentrations of 1 µM, 5 µM, and 10 µM, compound 2-amino-4-fluorobenzoic acid had no statistically significant effect on the growth of *Ralstonia solanacearum*. Data are expressed as mean ± standard deviation and are representative results of three independent experiments. ns indicates no significant difference (P > 0.05). Statistical comparisons were performed using one-way ANOVA. This indicates that at the highest concentration used (10 µM), the inhibitory effect of 2-amino-4-fluorobenzoic acid on the quorum sensing of *Ralstonia solanacearum* is not achieved by inhibiting its growth.

[0042] Example 3: Biological control and survival rate of 2-amino-4-fluorobenzoic acid against bacterial wilt in tomato

[0043] Five treatment groups were set up: no tomato plants as the control group, GMI1000 group, GMI1000+1 µM 2A4FBA group, GMI1000+5 µM 2A4FBA group, and GMI1000+10 µM 2A4FBA group. Each treatment was replicated six times, and one tomato plant was planted per pot. Seven days after planting, inoculation was carried out by root irrigation. The OD of the Ralstonia solanacearum inoculum solution was... 600 The inoculum was adjusted to version 1.0. All pots were placed in a greenhouse environment with daytime temperatures of 25-28℃ and nighttime temperatures of 20-25℃, with 12 hours of light per day. The position of the potted plants was randomly adjusted every two days to minimize environmental interference, and the time and number of dead tomato plants in each pot were recorded. Photos were taken on the 14th day after inoculation. Figure 3 and Figure 4 It was found that most tomato plants in the group treated with Ralstonia solanacearum died from the disease, while the group treated with 2-amino-4-fluorobenzoic acid showed a significant reduction in disease incidence. This indicates that 2-amino-4-fluorobenzoic acid can effectively reduce the severity of bacterial wilt in tomatoes and decrease the mortality rate of tomato plants.

[0044] Example 4: 2-Amino-4-fluorobenzoic acid-induced Ralstonia solanacearum colony counts in tomato roots and stems

[0045] Fourteen days after inoculation, three tomato seedlings were taken from each experimental group. The soil around the roots was washed away with sterile water, and the seedlings were blotted dry with sterile paper. One g each of tomato root and stem were weighed using an analytical balance, ground in a sterile mortar in a laminar flow hood, and then 6 mL of sterile water was added and transferred to a sterile centrifuge tube. After serial dilution, 100 µL was spread onto CPG plates, dried in a laminar flow hood, and then sealed. The plates were incubated upside down in a 28°C incubator for 3 days. The colony count (CFU) on the plates was then recorded. The colony count of *Ralstonia solanacearum* on the tomato roots and stems represents the colonization ability of *Ralstonia solanacearum* in the tomato plant roots and stems. Figure 5 Figures A and B show that the number of Ralstonia solanacearum colonies in the treatment group treated with 2-amino-4-fluorobenzoic acid and simultaneously inoculated with Ralstonia solanacearum was significantly less than that in the control group inoculated with Ralstonia solanacearum alone, indicating that 2-amino-4-fluorobenzoic acid can significantly inhibit the growth and reproduction of Ralstonia solanacearum in tomato plants.

[0046] Example 5: Expression and purification of RaaR protein

[0047] The designed expression primer pair for the RaaR gene is as follows:

[0048] Upstream primer: 5'-CCG GAATTC ATGGACTTGCGAAAACTGCC-3' (underlined is the EcoRI restriction site), sequence as shown in SEQ ID NO.3;

[0049] Downstream primer: 5'-CCC AAGCTT CTATGCCGCGCGGCCGG-3' (underlined is the HindIII restriction site), sequence as shown in SEQ ID NO.4;

[0050] The RaaR gene coding region was amplified using primers extracted from Ralstonia solanacearum GMI1000 genomic DNA as a template. The reaction system was 50 µL: 2 µL DNA template, 2 µL each of forward and reverse primers, 25 µL 2×Taq HiFi PCR mix (product number: XKL0211), and 19 µL ddH2O. The PCR conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 30 s, 57℃ annealing for 30 s, and 72℃ extension for 45 s, for 35 cycles; followed by a 72℃ extension for 10 min. The pMAL-c5X expression vector was digested with HindIII and EcoRI restriction sites. The digestion system was 20 µL: 10 µL pMAL-c5X expression vector, 1 µL EcoRI enzyme (Catalog No.: R3104S; New England Biolabs), 1 µL HindIII enzyme (Catalog No.: D6330-1; Beyotime), 2 µL rCutSmartBuffer (Catalog No.: B6004S; New England Biolabs), and 6 µL ddH2O. The digestion conditions were: 37℃ for 3 h, followed by inactivation at 65℃ for 20 min. The enzyme was then ligated to the vector, and the recombinant plasmid was constructed (structural diagram shown). Figure 10 (As shown) The bacteria were transferred into Escherichia coli BL21 and screened on a plate containing ampicillin. Single colonies were picked and inoculated into LB liquid medium, ampicillin was added, and the culture was incubated at 37°C until OD200. 600 =0.6-0.8, add 1 mM IPTG and induce expression at 16℃. Centrifuge to remove supernatant, add 1×PBS buffer to bacterial cells, resuspend, sonicate, and collect supernatant by centrifugation. Affinity purification is performed using an MBP affinity chromatography column, followed by elution with 1×PBS buffer. Finally, protein purity and concentration are verified by SDS-PAGE using a 10-180 kDa pre-stained protein marker, and stored at -80℃. Figure 6 As shown in Figure A, the RaaR protein obtained through expression and purification has high purity and can be used for subsequent ITC experiments. The expressed RaaR protein is a fusion protein fused with the MBP tag, so the purified protein size is 76 kDa. The RaaR amino acid sequence is shown in SEQ ID NO.1. The MBP amino acid sequence is shown in SEQ ID NO.2.

[0051] SEQ ID NO.1:

[0052] MDLRKLPNLGALKMFEAAARLESFSRAAEALCVTHSAVSHQIRALEAELGMVLFKRDGRRITLTDPGRRYAERIRSALFEIAAATESMRTGDRERRLVISLLPSFAARWLTPRIGRFIERYPEIDVELQSTHVITDFNRDDVDVVLRLSDGNDPDLFVEPLLDEVFFPACAPTFNGGRLPKTPADLADVTLLRSDYEMWRAWFMAAGLEGWAEPRRGVLYQDASQLMQASMEGQGVGLVRRSLAMQEIINGRLVRLFDIDAPSPWTYWFICPQPLLDTFRVRALREWLHEEVAQFRALYERSPTAVTMAGRAA。

[0053] SEQ ID NO.2:

[0054] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT。

[0055] Example 6: ITC detection of the binding of RaaR and 2-amino-4-fluorobenzoic acid

[0056] In an ITC-200 microcalorimeter, 350 µL of a 20 µM RaaR protein solution was added to the sample cell, and the protein was titrated with 40 µL of 200 µM 2-amino-4-fluorobenzoic acid. This was followed by 19 consecutive 2 µL injections. A PBS buffer control group was included; 350 µL of PBS buffer was slowly added to the sample cell using a sampling needle, and then 40 µL of 200 µM 2-amino-4-fluorobenzoic acid was added for titration. The experiment was repeated three times, and the titration curves were fitted using MicroCal ORIGIN (v.7) software to determine the binding constant, thus fully verifying the binding relationship between RaaR protein and 2-amino-4-fluorobenzoic acid. Figure 6 As shown in Figure B, the Kd value of RaaR protein and 2-amino-4-fluorobenzoic acid is 0.738±0.249 µM, which indicates that 2-amino-4-fluorobenzoic acid can bind to RaaR protein. This also shows that RaaR is the target of 2-amino-4-fluorobenzoic acid in inhibiting the virulence of Ralstonia solanacearum.

[0057] Example 7: Biological control and survival rate of tomato bacterial wilt by combined application of 2-amino-4-fluorobenzoic acid and bacteriophage PN12-4

[0058] The publication number of the patent application related to the Ralstonia solanacearum phage PN12-4 used is CN 118813554 A. Six treatment groups were set up: tomato plants without any treatment as a blank group, PN12-4 group, GMI1000 group, GMI1000 and PN12-4 combined with 1 µM 2A4FBA group (GMI1000+PN12-4+1 µM 2A4FBA), GMI1000 and PN12-4 combined with 5 µM 2A4FBA group (GMI1000+PN12-4+5 µM 2A4FBA), and GMI1000 and PN12-4 combined with 10 µM 2A4FBA group (GMI1000+PN12-4+10 µM 2A4FBA). Each treatment group was set up with 3 replicates, and 2 tomato plants were planted in each pot. Seven days after planting, inoculation was carried out using the root irrigation method with OD of Ralstonia solanacearum GMI1000 bacterial solution. 600 Adjust to 1.0. During inoculation, add 10 µL of phage to 990 µL of bacterial culture to achieve a phage concentration of 10. 4 PFU / mL. PN12-4 group: Inoculated only in tomato plants at a concentration of 10. 4 PFU / mL phage; GMI1000 group: inoculated only with OD 600 =1.0 GMI1000 bacterial culture; GMI1000 and PN12-4 combined with 1 µM 2A4FBA group: GMI1000 bacterial culture OD600 When the concentration of 2A4FBA reaches 0.01, add 1 µM 2A4FBA to a final concentration and incubate until the bacterial OD of the culture reaches zero. 600 After reaching a concentration of 1.0, 990 µL of bacterial suspension was added to 10 µL of bacteriophage and inoculated into tomato plants. The same treatment was applied to the groups treated with GMI1000 and PN12-4 with 5 µM 2A4FBA, as well as the groups treated with GMI1000 and PN12-4 with 10 µM 2A4FBA, except for the final concentration of 2-amino-4-fluorobenzoic acid. When the tomato plants had 2-3 leaves, all six treatments were inoculated at the same time. The time and number of dead plants were recorded, and photographs were taken on day 14 post-inoculation. Figure 7 and Figure 8 As shown, most tomato plants in the single-inoculation group with Ralstonia solanacearum (Syndrome Azotobacterium wilt) developed the disease and died. However, in the group treated with a combination of 2-amino-4-fluorobenzoic acid and bacteriophage PN12-4, simultaneously inoculated with Ralstonia solanacearum, the disease incidence was significantly reduced. The lower concentration of bacteriophage used during inoculation indicates that the combined use of 2-amino-4-fluorobenzoic acid and low-concentration bacteriophage PN12-4 enhances the tomato plants' defense against Ralstonia solanacearum and reduces the mortality rate of tomato plants infected with the disease. Furthermore, compared to the use of 2-amino-4-fluorobenzoic acid alone, the combined use of the two showed a better synergistic control effect.

[0059] Example 8: Determination of Ralstonia solanacearum colony count in tomato roots and stems using a combination of 2-amino-4-fluorobenzoic acid and bacteriophage.

[0060] Fourteen days after inoculation, three tomato seedlings were taken from each experimental group. The soil around the roots was washed away with sterile water, and the seedlings were blotted dry with sterile paper. One g each of tomato root and stem were weighed using an analytical balance, ground in a sterile mortar in a laminar flow hood, and then 6 mL of sterile water was added and transferred to a sterile centrifuge tube. After serial dilution, 100 µL was spread onto CPG plates, dried in a laminar flow hood, and then sealed. The plates were incubated upside down in a 28°C incubator for 3 days. The colony count (CFU) on the plates was then recorded. The colony count of *Ralstonia solanacearum* on the tomato roots and stems represents the colonization ability of *Ralstonia solanacearum* in the tomato plant roots and stems. Figure 9 As shown in Figures A and B, the number of Ralstonia solanacearum colonies in the treatment group treated with a combination of 2-amino-4-fluorobenzoic acid and bacteriophage PN12-4, along with simultaneous inoculation with Ralstonia solanacearum, was significantly lower than that in the control group inoculated with Ralstonia solanacearum alone. This indicates that the combined use of the two treatments can significantly inhibit the growth and reproduction of Ralstonia solanacearum in tomato plants. Furthermore, compared to the use of 2-amino-4-fluorobenzoic acid alone, the combined use of the two treatments resulted in fewer Ralstonia solanacearum colonies in the roots and stems of tomato plants, demonstrating a more significant inhibitory effect on Ralstonia solanacearum.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A RaaR protein inhibitor of Ralstonia solanacearum, characterized in that, The Ralstonia solanacearum RaaR protein inhibitor is a 2-amino-4-fluorobenzoic acid compound.

2. The use of the Ralstonia solanacearum RaaR protein inhibitor according to claim 1 in the preparation of anti-Ralstonia solanacearum drugs.

3. A fungicide for treating bacterial wilt, characterized in that, Including the Ralstonia solanacearum RaaR protein inhibitor as described in claim 1.

4. The antibacterial agent according to claim 3, characterized in that, It also includes Ralstonia solanacearum phage PN12-4.

5. The antibacterial agent according to claim 3, characterized in that, The concentration of the Ralstonia solanacearum RaaR protein inhibitor is not less than 1 μM.

6. The antibacterial agent according to claim 5, characterized in that, The concentration of the Ralstonia solanacearum RaaR protein inhibitor is 1 μM, 5 μM or 10 μM.

7. The antibacterial agent according to claim 4, characterized in that, The titer of the Ralstonia solanacearum phage PN12-4 is not less than 10. 4 PFU / mL.

8. The antibacterial agent according to claim 7, characterized in that, The titer of the Ralstonia solanacearum phage PN12-4 is 10. 4 PFU / mL.

9. The antibacterial agent according to claim 3, characterized in that, The antibacterial agent is in the form of an aqueous solution.

10. The application of the antibacterial wilt agent according to any one of claims 3-9 in tomato plants.

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