Application of pear fire blight bacterium effect factor HrpG in prevention and control of pear fire blight

Abstract

The invention discloses an application of a pear fire blight bacterium effect factor HrpG in prevention and control of pear fire blight. A nucleotide sequence of the effect factor HrpG gene is shown as SEQ ID NO: 1. Through a gene editing technology, the pyraclospermum palmatum mutant delta HrpG of which the effector HrpG gene is knocked out is constructed. Korla young fruits and pyrus betulaefolia in-vitro leaves are inoculated through a control group of wild fire blight bacteria and delta HrpG, and the delta HrpG is found to be slower in morbidity and smaller in scabs; the mutant delta HrpG is applied to plants or a plant growth environment, and biological prevention and control of fire blight are achieved through the competitive effect of the mutant delta HrpG and wild type pathogenic bacteria. Therefore, delta HrpG is weaker in pathogenicity, and provides a new strategy for green prevention and control of pear fire blight in application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to application of an effector HrpG of Erwinia amylovora in preventing and controlling pear fire blight. BACKGROUND

[0002] Pear trees are important economic fruit trees widely cultivated in the world, and their fruits are loved by consumers due to rich nutrition and unique flavor, and have significant economic value. However, in many production areas around the world, pear trees are often seriously threatened by devastating diseases such as fire blight, which directly affects the growth and development of trees, fruit quality and yield. Fire blight is caused by Erwinia amylovora, and its occurrence is closely related to regional climate conditions. Warm and humid environment is particularly conducive to the spread and infection of the pathogen, and rainy or high-humidity weather during flowering and young fruit stage often leads to the outbreak and prevalence of the disease.

[0003] Pear fire blight is a devastating bacterial disease caused by Erwinia amylovora, which causes serious economic losses in many pear production areas around the world. The pathogen can infect various tissues of pear trees, causing symptoms such as fire-like wilting and blackening of flowers, leaves, branches and fruits, and even causing the death of the whole plant. At present, the prevention and control of the disease mainly rely on antibiotics and copper preparations, but long-term use can easily cause problems such as drug resistance of the pathogen, environmental pollution and pesticide residues, and the prevention and control effect is often restricted by climate conditions. Therefore, developing new, efficient and environmentally friendly sustainable prevention and control strategies has become an urgent need for the healthy development of pear industry.

[0004] The pathogenicity of Erwinia amylovora mainly depends on the type III secretion system (T3SS), which can inject a series of effector proteins (effectors) into host cells to interfere with the immune response of plants, thereby successfully colonizing and causing disease. Traditional researches are mainly focused on the function of effectors themselves, and the application of precisely targeting and interfering with the upstream key genes of the regulation network to fundamentally undermine the pathogenicity of the pathogen is still insufficient.

[0005] There is a lack of effective means for preventing and controlling the disease by directly targeting the key regulatory genes of the pathogenicity of pear fire blight in the prior art. It is of great significance to develop new genetic engineering biological pesticides, cultivate disease-resistant varieties and develop new prevention and control technical schemes to further clarify the function of the gene and develop its application as a molecular target in biological control. SUMMARY

[0006] The purpose of the present application is to provide a virulent effector HrpG of pear fire blight and its application in the prevention and control of pear fire blight.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0008] In a first aspect, the present application claims at least one use of a nucleotide sequence as shown in SEQ ID NO: 1 of an effector HrpG gene in at least one of (a1)-(a2):

[0009] (a1) preventing and controlling bacterial wilt of plants;

[0010] (a2) preparing a product for preventing and controlling bacterial wilt of plants.

[0011] In a second aspect, the present application claims at least one use of a biological material related to a nucleotide sequence as shown in SEQ ID NO: 1 of an effector HrpG gene in at least one of (a1)-(a2):

[0012] (a1) preventing and controlling bacterial wilt of plants;

[0013] (a2) preparing a product for preventing and controlling bacterial wilt of plants;

[0014] The biological material related to the effector HrpG gene is a protein encoded by the effector HrpG gene or a biological material for inhibiting or knocking out the effector HrpG gene;

[0015] The biological material for inhibiting or knocking out the effector HrpG gene is at least one of (b1)-(b3):

[0016] (b1) a nucleic acid molecule for inhibiting or knocking out the effector HrpG gene;

[0017] (b2) a recombinant vector containing the nucleic acid molecule of (b1);

[0018] (b3) a recombinant microorganism containing the nucleic acid molecule of (b1) or a recombinant microorganism containing the recombinant vector of (b2).

[0019] Further, the protein encoded by the effector HrpG gene is a protein as shown in (c1) or (c2):

[0020] (c1) a protein having an amino acid sequence as shown in SEQ ID NO: 2;

[0021] (c2) a fusion protein having the same function obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein of (c1).

[0022] Further, the use is at least one of (d1)-(d2):

[0023] (d1) weakening the pathogenicity of a bacterial wilt pathogen by inhibiting or knocking out the effector HrpG gene;

[0024] (d2) constructing a Xanthomonas campestris pathogenic bacteria mutant AHrpG with the effect factor HrpG gene knocked out, and applying the mutant AHrpG to plants or plant growth environment, and realizing biological control of the bacterial wilt of plants through competition between the mutant AHrpG and wild type pathogenic bacteria.

[0025] Further, the effect factor HrpG gene is inhibited or knocked out by targeting the effect factor HrpG gene or the protein encoded by the effect factor HrpG gene using a gene editing technology, an RNA interference technology or a chemical inhibitor.

[0026] In a third aspect, the present application claims a method for preventing and controlling bacterial wilt, which is at least one of the following (d1) and (d2):

[0027] (d1) weakening the pathogenicity of Xanthomonas campestris pathogenic bacteria by inhibiting or knocking out the effect factor HrpG gene with the nucleotide sequence as shown in SEQ ID NO: 1;

[0028] (d2) constructing a Xanthomonas campestris pathogenic bacteria mutant AHrpG with the effect factor HrpG gene knocked out, and applying the mutant AHrpG to plants or plant growth environment, and realizing biological control of the bacterial wilt of plants through competition between the mutant AHrpG and wild type pathogenic bacteria.

[0029] In a fourth aspect, the present application claims a Xanthomonas campestris pathogenic bacteria mutant AHrpG with the effect factor HrpG gene knocked out, which is constructed by the following steps:

[0030] (1) amplifying the upstream homologous arm sequence as shown in SEQ ID NO: 4, the downstream homologous arm sequence as shown in SEQ ID NO: 5 and the gentamicin resistance gene sequence as shown in SEQ ID NO: 3 of the effect factor HrpG gene;

[0031] (2) connecting the upstream homologous arm sequence, the gentamicin resistance gene sequence and the downstream homologous arm sequence to form a LF-GM-RF fusion fragment;

[0032] (3) cloning the LF-GM-RF fusion fragment into a suicide plasmid pK18mobsacB to construct a recombinant plasmid;

[0033] (4) introducing the recombinant plasmid into Xanthomonas campestris pathogenic bacteria through conjugation transfer, and obtaining the Xanthomonas campestris pathogenic bacteria mutant AHrpG with the effect factor HrpG gene knocked out through twice homologous recombination screening.

[0034] Fifthly, this invention claims protection for a biocontrol agent for fire blight, the agent comprising a fire blight pathogen mutant ΔHrpG with the HrpG gene knocked out, as shown in SEQ ID NO:1. Further, the mutant ΔHrpG is the fire blight pathogen mutant ΔHrpG constructed using the aforementioned construction process.

[0035] In the technical solution of this invention, the pathogen of fire blight is Erwinia amylovora.

[0036] In the technical solution of this invention, the plant is selected from pear species (e.g., pear) or model plants used for pathogenicity identification (e.g., tobacco); the pear is Korla fragrant pear (Pyrus sinkiangensis) or wild pear (Pyrus betulifolia), and the tobacco is Nicotiana benthamiana or large-leaved tobacco (Nicotiana atabacum).

[0037] The HrpG gene, an effector of the present invention, was obtained by PCR cloning using Erwinia amylovora DNA as a template. Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2.

[0038] Prior to this invention, there were no publicly reported tests on the pathogenicity of the effector factor HrpG in pears. This invention is the first to knock out this gene and elucidate the timing and severity of disease onset after pear inoculation following knockout.

[0039] This invention constructs a plant overexpression vector containing the HrpG gene, a potentiogenic factor for pear fire blight virus, and expresses this effector in *Nicotiana benthamiana* using an Agrobacterium-mediated gene expression system, which elicits a hypersensitive cell necrosis response. Tobacco exhibits disease resistance and simultaneously induces localized programmed cell death (PCD) near the infection site to inhibit pathogen spread, i.e., a hypersensitive response (HR).

[0040] The identification process of the pathogenicity of the pear fire blight virus virulence effector factor HrpG in this invention includes the following steps:

[0041] (1) Construction and acquisition of the overexpression structure of the effect factor HrpG: The HrpG open reading frame was cloned into a plant expression vector and expressed under the drive of a strong promoter;

[0042] (2) Obtaining the HrpG overexpression structure from Agrobacterium: The constructed HrpG overexpression structure was transformed into Agrobacterium competent cells by freeze-thaw method;

[0043] (3) Pathogenicity identification of effect factor HrpG: HrpG is expressed in tobacco styrax, and the activation of allergic cell necrosis reaction is observed to determine the pathogenicity of effect factor HrpG.

[0044] This invention constructed a *Erwinia amylase* material ΔHrpG with the effector gene HrpG knocked out. Based on the pathogenicity assessment of the effector gene HrpG, *Erwinia amylase* (WT) and the *Erwinia amylase* with HrpG knocked out (ΔHrpG) were inoculated into young Korla fragrant pear fruits and detached *Pyrus pyrifolia* leaves, respectively, and the time of disease onset and lesion area were observed. Simultaneously, inoculation experiments were conducted on *Nicotiana benthamiana* and *Nicotiana macrophylla*. Disease incidence was detected, and the function of the effector gene HrpG was verified (e.g.,...). Figure 1 (As shown).

[0045] The beneficial effects of this invention are:

[0046] Significantly Reduced Pathogenicity: This invention utilizes gene editing technology to construct the *Phytophthora infestans* material ΔHrpG, in which the effector factor HrpG is knocked out. Knockout of the HrpG gene directly prevents the *Phytophthora infestans* from effectively infecting parasitic plants. Inoculation of young fruits from Korla and detached leaves of *Pyrus pyrifolia* with the control group wild-type *Phytophthora infestans* using ΔHrpG revealed that ΔHrpG resulted in slower disease development and smaller lesions. This directly demonstrates the crucial role of HrpG as a virulence factor, and targeting it can effectively inhibit disease development.

[0047] In line with the green prevention and control and pesticide reduction and efficiency enhancement strategy: This invention provides a precise prevention and control strategy based on the pathogenic mechanism of pathogens, which is expected to reduce the use of traditional broad-spectrum antibiotics (such as streptomycin), help delay the development of drug resistance, reduce pesticide residues, protect the ecological environment and food safety, and fully comply with the direction of sustainable agricultural development in my country and even the world.

[0048] The control strategy is novel and highly sustainable: Unlike the traditional "sterilization" approach, this strategy controls disease by "eliminating toxins," altering the interaction between pathogens and hosts. This "using bacteria to treat bacteria" or "targeted disease control" approach is more advanced and sustainable, and less likely to induce simple drug resistance mutations in pathogens. Attached Figure Description

[0049] Figure 1 This is a technical flowchart of the present invention.

[0050] Figure 2These are agarose gel electrophoresis images of PCR identification after knocking out the effector factor HrpG. A shows the electrophoresis images of cloning the effector factor HrpG simultaneously in WT colonies and the knockout colony (ΔHrpG); M: DL2000 marker; 1-6: Knockout colony (ΔHrpG); the gel images show that the knockout colony (ΔHrpG) cannot clone the HrpG sequence. B shows the electrophoresis images of cloning the sequence between the upper and lower homologous arms of the effector factor HrpG simultaneously in WT colonies and the knockout colony (ΔHrpG); M: DL2000 marker; 1-6: Knockout colony (ΔHrpG); since HrpG is replaced by a gentamicin gene sequence after knockout, this gentamicin sequence is 353 bp longer than the HrpG sequence; the dashed line indicates a 353 bp difference in the bands of the two colony clones.

[0051] Figure 3 The images show the growth of knockout colonies (ΔHrpG) and WT colonies after 24 h of incubation on a plate at 28 ℃. The growth of knockout colonies (ΔHrpG) was not significantly different from that of WT colonies, indicating that the knockout effector HrpG does not affect the growth of pear fire blight pathogen.

[0052] Figure 4 The study aimed to elicit a hypersensitive necrosis response in plants 48 h after the expression of the effector HrpG in Nicotiana benthamiana. Specifically, 1) pCAMBIA1300-HrpG could elicit a hypersensitive necrosis response; 2) the positive control pCAMBIA1300-INF1 could also elicit a hypersensitive necrosis response; and 3) the empty vector pCAMBIA1300 did not induce a hypersensitive necrosis response in plants.

[0053] Figure 5 This chart shows the disease incidence of young fruits in Korla simultaneously inoculated with WT and ΔHrpG at different time points. A represents the disease incidence 24 hours after inoculation; B represents 48 hours after inoculation; C represents 72 hours after inoculation; D represents 96 hours after inoculation; E represents 120 hours after inoculation; F represents 144 hours after inoculation; and G is an analysis of lesion area data for the six time points after inoculation. The results show that the knockout colonies exhibited significantly reduced pathogenicity, smaller lesion areas, and slower disease progression.

[0054] Figure 6 This is a diagram showing the disease progression of detached pear leaves simultaneously inoculated with WT and ΔHrpG.

[0055] Figure 7 This is a diagram showing the disease incidence of both large-leaf tobacco and Benzodiaceae tobacco when simultaneously inoculated with WT and ΔHrpG.

[0056] Figure 8This is a diagram showing the results of an experiment on the competitive interaction between WT bacterial solution and ΔHrpG bacterial solution in young Korla fragrant pears. Detailed Implementation

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0058] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0060] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0061] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0062] The room temperature described in this invention is 25±5℃.

[0063] Example 1 Cloning of the effector factor HrpG and construction of its overexpression vector

[0064] 1. Extraction of Erwinia amyloliquefaciens genomic DNA

[0065] The *Erwinia amyloliquefaciens* strain used in this study was provided by the College of Life Sciences, Nanjing Agricultural University. The tested pear fire blight strain was inoculated onto NA (beef extract 3 g / L, peptone 10 g / L, sodium chloride 5 g / L, pH 7.0-7.4; sterilized at 115 ℃ for 30 min) solid medium and activated by incubation at 28 ℃ for 24 h. Single colonies were picked and cultured in NA liquid medium at 28 ℃ with shaking at 180 r / min for 12 h. DNA was extracted using the conventional CTAB method. The specific steps are as follows:

[0066] (1) Place a small amount of quartz sand into a 1.5 mL centrifuge tube, cut off the tip of a 1000 μL pipette tip, add 500 μL of bacterial solution to the centrifuge tube, and grind at 60 Hz for 120 s.

[0067] (2) Add 700 μL of CTAB extract and 10 μL of mercaptoethanol, mix slowly, and incubate in a metal bath at 65 °C for 1 h.

[0068] (3) Centrifugation: 10,000 rpm, 25 ℃, 10 min. Transfer the supernatant to a new tube, add 700 μL of chloroform, mix slowly up and down for 5 min, and let stand on ice for 10 min.

[0069] (4) Centrifugation: 13000 rpm, 25 ℃, 7 min. Transfer the supernatant to a new tube, add 400 μL of isopropanol, mix well, and let stand at -20 ℃ for 30 min.

[0070] (5) Centrifugation: 12000 rpm, 25 ℃, 7 min. Discard the supernatant, add 1 mL of 75% ethanol, and gently wash the precipitate.

[0071] (6) Centrifugation: 10000 rpm, 25 ℃, 3 min. Discard the supernatant, open the tube and air dry, dissolve the precipitate with 30 μL dd H2O (add 1 μL RNase A), and store the obtained DNA at -20 ℃.

[0072] 2. Amplification of effector factors

[0073] Forward primer: aacacgggggactctagaATGAGATCAACTGAATTG (SEQ ID NO:6)

[0074] Reverse primer: ttgctcaccatggatccACCATGGTTTCCCC (SEQ ID NO:7)

[0075] The PCR amplification system consisted of: 1 μL template DNA, 20 μL dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL nuclease-free water.

[0076] PCR was performed according to the following procedure: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, 40 cycles, followed by a 72 °C extension for 10 min after each cycle.

[0077] The full-length sequence was amplified using a high-fidelity enzyme, and the PCR product was then purified and recovered by gel cutting after 1% agarose gel electrophoresis.

[0078] 3. Construction of overexpression vectors

[0079] The overexpression vector pCAMBIA1300 plasmid was digested with Xba I and BamHI. The amplified target gene was then ligated into the digested pCAMBIA1300 vector via homologous recombination. The ligation product was transformed into competent E. coli DH5α cells using a heat shock method. The effector gene HrpG was successfully cloned through kanamycin plate screening, PCR identification, and sequencing. The overexpression structure pCAMBIA1300-HrpG was obtained. The double digestion system of the overexpression vector is shown in Table 1, and the ligation system is shown in Table 2.

[0080] Table 1 Double enzyme digestion system

[0081]

[0082] Table 2 Connection System

[0083]

[0084] The correctly sequenced pCAMBIA1300-HrpG recombinant vector was transfected into Agrobacterium competent cells (GV3101) using a freeze-thaw method and stored at -80 °C for subsequent in vivo expression in tobacco.

[0085] Example 2: Identification of the pathogenicity of HrpG by tobacco hypersensitivity reactions induced by the effector factor HrpG.

[0086] The preserved Agrobacterium GV3101 was activated and cultured. Single colonies were selected and cultured in small quantities. The culture was then incubated overnight at 220 rpm at 28 °C. 4 mL of culture medium was then incubated at a 1:10 volume ratio of bacterial culture to liquid culture medium. The culture was then incubated at 220 rpm for 4 h. The OD of the bacterial culture was measured. 600Centrifuge at 5000 rpm for 8 min at room temperature, maintaining an OD value of 0.6–0.8. Resuspend the bacterial culture in resuspending buffer (1 mL of 10 M MgCl2, 1 mL of 10 M MES, 75 μL of 200 mM acetylsylphenone, and bring the total volume to 100 mL with ddH2O) until the OD value is reached. 600 The concentration was 0.5, and the cell culture was allowed to recover at 28 °C for 1.5 h. A pair of positive control groups were also set up, including *Agrobacterium tumefaciens* GV3101 containing the pCAMBIA1300-INF1 plasmid (INF1 acts as a positive control to ensure the validity of the immunization experiment by mimicking two key signaling pathways of T cell activation) and *Agrobacterium tumefaciens* GV3101 containing the empty vector pCAMBIA1300 plasmid.

[0087] Aspirate the bacterial solution with a 1 mL sterile syringe, remove the needle, and immerse the solution on the back of the tobacco. Mark the immersion area by drawing a circle. Observe whether it can induce allergic cell necrosis after 48 hours.

[0088] The in vivo analysis results of tobacco in this embodiment show that the effector factor HrpG of Erwinia amylase can induce hypersensitive cell necrosis. Figure 4 Meanwhile, the positive control pCAMBIA1300-INF1 could also induce hypersensitive cell necrosis, and Agrobacterium GV3101 containing the empty vector pCAMBIA1300 could not induce hypersensitive cell necrosis. This indicates that the effector factor HrpG has strong pathogenicity.

[0089] Example 3: Knockout of the effector factor HrpG

[0090] 1. Clones containing the gentamicin (GM) gene sequence

[0091] The gentamicin (GM) gene is a gene sequence containing gentamicin resistance. This sequence is subsequently used to replace the effector factor HrpG, resulting in gentamicin-resistant colonies. Further selection is performed using plates containing gentamicin-resistant colonies. The nucleotide sequence of the gentamicin (GM) gene is shown in SEQ ID NO:3. The primers used to amplify the gentamicin (GM) gene sequence are shown below:

[0092] Forward primer: TTGCCAAAGCCATCATCAggaaacggatgaaggcacgaa (SEQ ID NO:8)

[0093] Reverse primer: AGCGCAACTCTCGTTTCacgaattgttaggtggcg (SEQ ID NO:9)

[0094] The PCR amplification system consisted of: 1 μL template containing GM, 20 μL dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL nuclease-free water.

[0095] The PCR program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, for 40 cycles, followed by a 72 °C extension for 10 min after each cycle. The GM gene PCR product was then purified and recovered by 1% agarose gel electrophoresis and gel cutting.

[0096] 2. Upstream homologous arm gene (LF) and downstream homologous arm gene (RF) of the cloning effector HrpG.

[0097] The nucleotide sequence of the upstream homologous arm gene (LF) of the effector factor HrpG is shown in SEQ ID NO:4, and the nucleotide sequence of the downstream homologous arm gene (RF) of the effector factor HrpG is shown in SEQ ID NO:5. Primers used for amplifying the upstream and downstream homologous arms are shown below:

[0098] Forward primer of upstream homologous arm (LF): tgatttagtgtatgatggtGCTGGCAGCAGCAATATCAGC (SEQ ID NO:10)

[0099] Upstream homologous arm (LF) reverse primer: ttcgtgccttcatccgtttccTGATGATGGCTTTGGCAAG (SEQ ID NO:11)

[0100] Downstream homologous arm (RF) forward primer: ccacctaacaattcgtGAAACGAGAGTTGCGCTGCCGG (SEQ ID NO: 12)

[0101] Downstream homologous arm (RF) reverse primer: actggagcacctcaaaaacTTTACCGTTCATCAGCTCATT (SEQ ID NO:13)

[0102] The PCR amplification system consisted of: 1 μL of Erwinia amyloliquefaciens DNA template, 20 μL of dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL of nuclease-free water.

[0103] The PCR program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, 40 cycles, followed by a 72 °C extension for 10 min after each cycle. The PCR products of the LF and RF genes were then purified and recovered by 1% agarose gel electrophoresis followed by gel cutting.

[0104] 3. Connection of upper and lower homologous arms to the GM gene

[0105] Using the PCR product purified and recovered via gel in steps 1 and 2 above as a template, the upstream homologous arm (LF), the GM gene, and the downstream homologous arm (RF) are connected by the forward primer of the upstream homologous arm and the reverse primer of the downstream homologous arm. This connection is referred to as the LF-GM-RF gene.

[0106] The PCR amplification system consisted of: 1 μL of gel-recovered product as template, 20 μL of dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL of nuclease-free water.

[0107] The PCR program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, for 40 cycles, followed by a 72 °C extension for 10 min after each cycle. The LF-GM-RF gene PCR product was then purified and recovered by 1% agarose gel electrophoresis and gel cutting.

[0108] 4. Linearize the suicide plasmid pK18mobsacB.

[0109] Suicide plasmids operate on the principle of being unable to replicate within host cells. They are typically derivatives of R plasmids and have a broad host range. Their replication requires a specific protein, which most bacteria do not produce. Therefore, when a suicide plasmid enters a host cell, it either fails to replicate and is eliminated, or it integrates into the host's chromosome, replicating along with it. By cloning the mutated target gene into a suicide plasmid vector, secondary homologous recombination can be achieved in the host, producing mutant strains with the mutation. pK18mobsacB (purchased from Shanghai Newp Biotechnology Co., Ltd., plasmid number: V005269) is one such example, designed to integrate the LF-GM-RF gene into the chromosome of Erwinia amyloliquefaciens.

[0110] The circular plasmid pK18mobsacB was amplified into a linear plasmid by designing a pair of primers.

[0111] Forward primer: GCTGATATTGCTGCTGCCAGCaccatcatacactaaatca (SEQ ID NO:14)

[0112] Reverse primer: AATGAGCTGATGAACGGTAAAgtttttgaggtgctccagt (SEQ ID NO:15)

[0113] The PCR amplification system consisted of: 1 μL pK18mobsacB plasmid as template, 20 μL dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL nuclease-free water.

[0114] The PCR program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, for 40 cycles, followed by a 72 °C extension for 10 min after each cycle. The PCR product of the linear pK18mobsacB gene was then purified and recovered by 1% agarose gel electrophoresis and gel cutting.

[0115] 5. Homologous recombination of the LF-GM-RF gene with linear pK18mobsacB was performed to form a circular plasmid.

[0116] The ligation product was transformed into competent E. coli DH5α cells using the heat shock method. After plate screening with kanamycin and gentamicin, PCR identification, and sequencing identification by a company, the pK18mobsacB plasmid carrying the LF-GM-RF gene was successfully constructed, hereinafter referred to as pK18-LGR.

[0117] 6. The suicide plasmid pK18-LGR was introduced into Erwinia amyloliquefaciens using the helper plasmid pRk600.

[0118] *E. coli* carrying pRk600 plasmid (purchased from the BioVector Plasmid Vector Strains Cell Protein Antibody Gene Preservation Center) and pK18-LGR plasmid were inoculated onto LB solid medium and cultured at 36 °C for 24 h to activate. Single colonies were picked and cultured in LB liquid medium at 36 °C with shaking at 180 r / min for 12 h. Simultaneously, *Erwinia amyloliquefaciens* strains were inoculated onto NA solid medium and cultured at 28 °C for 24 h to activate. Single colonies were picked and cultured in NA liquid medium at 28 °C with shaking at 180 r / min for 12 h. The OD of the pK18-LGR bacterial culture was then measured. 600 pRk600 bacterial culture OD 600 : Erwinia amyloliquefaciens broth OD 600 When the ratio is 2:1:1 (v:v:v), the three bacterial cultures are mixed and inoculated onto NA solid medium.

[0119] The suicide plasmid pK18-LGR was introduced into Erwinia amyloliquefaciens using the helper plasmid pRk600, thereby integrating the LF-GM-RF gene into the chromosome of Erwinia amyloliquefaciens and replicating along with the chromosome.

[0120] 7. Screening and identification of knockout colonies

[0121] Single colonies grown on NA solid medium were picked and placed in NA liquid medium containing gentamicin, and cultured at 28°C with shaking at 180 r / min for 12 h. 100 μL of each colony was then plated onto gentamicin-resistant SBE (beef extract 8 g / L, sucrose 50 g / L, pH 7.0-7.4; sterilized at 115°C for 30 min) plates and incubated at 28°C for 24 h.

[0122] Method 1: Simultaneously select single colonies grown from Erwinia amyloliquefaciens on NA solid medium and SBE solid medium for PCR identification using a pair of primers. The purpose is to identify whether the colony contains the effector factor HrpG.

[0123] Forward primer: ATGAGATCAACTGAATTGCAGCAGT (SEQ ID NO:16)

[0124] Reverse primer: TCAACCATGGTTTCCCCTTGGTAAA (SEQ ID NO:17)

[0125] The PCR amplification system consisted of 1 μL of original colony (WT) and knockout colony (ΔHrpG) as templates, 20 μL of dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL of nuclease-free water.

[0126] PCR program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 40 cycles, followed by a 72℃ extension for 10 min after each cycle. Electrophoresis was then performed on a 1% agarose gel. Observation of the gel electrophoresis images showed that WT colonies exhibited bands the size of HrpG, while knockout colonies did not show bands the size of HrpG. Figure 2 (A in the middle).

[0127] Method 2: Simultaneously select single colonies grown from *Erwinia amyloliquefaciens* on NA solid medium and SBE solid medium for PCR identification, using a pair of primers. The aim is to determine whether the gene sequence between the upper and lower homologous arms of the colony differs by 353 bp from that of the original colony. This is because the replaced effector HrpG sequence differs from the GM gene sequence by 353 bp.

[0128] Forward primer: tgatttagtgtatgatggtGCTGGCAGCAGCAATATCAGC (SEQ ID NO:18)

[0129] Reverse primer: actggagcacctcaaaaacTTTACCGTTCATCAGCTCATT (SEQ ID NO:19)

[0130] The PCR amplification system consisted of 1 μL of original colony (WT) and knockout colony (ΔHrpG) as templates, 20 μL of dNTP Mix (10 mmol / L), 2 μL each of forward and reverse primers, and 20 μL of nuclease-free water.

[0131] PCR program completed: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 40 cycles, followed by a 72℃ extension for 10 min after each cycle. Electrophoresis was then performed on a 1% agarose gel. Observation of the gel electrophoresis images showed a 353 bp difference in band size between WT and HrpG amplification. Figure 2 (B in the middle).

[0132] Example 4: Detection of pathogenicity of knockout colonies (ΔHrpG)

[0133] Activation of the pathogen and preparation of inoculum: The tested pear fire blight strain (WT) and mutant strain (ΔHrpG) were cultured on NA solid medium and activated by incubation at 28 °C for 24 h (e.g., Figure 3 (As shown). Single colonies were picked and placed in NA liquid medium, and cultured at 28 °C with shaking at 180 r / min for 24 h. OD 600 A bacterial culture with a value of approximately 0.8 to 1.0 is used as the inoculum.

[0134] 1. Inoculation experiment of young fruit in Korla

[0135] Harvest healthy young Korla pears, with a diameter of 3-4 cm, and sterilize them by soaking them in 75% alcohol for 10 minutes. Use a 10 μL sterilization pipette tip to poke a hole about 1 cm in diameter on the surface of the young pear, and inoculate the hole with 10 μL of WT and ΔHrpG bacterial solution.

[0136] After inoculation, the young fruits were placed flat on trays lined with absorbent cotton, sealed with plastic wrap, and cultured in an artificial climate chamber with a photoperiod of L / D = 12 h / 12 ​​h, a temperature of 28 ℃, and a relative humidity of 75%. The time of bacterial ooze appearance and the size of the lesions were recorded.

[0137] By recording the lesion spread area at 24h, 48h, 72h, 96h, 120h, and 144h time intervals, the results showed that the pathogenicity of the knockout colonies was significantly reduced, the lesion area was smaller, and the disease progressed more slowly. Figure 5 ).

[0138] 2. In vitro leaf inoculation experiment of Pyrus pyrifolia

[0139] Healthy young leaves of *Pyrus pyrifolia* were collected from the greenhouse. The base was wrapped with sterile, water-soaked cotton wool, then sealed with plastic wrap to retain moisture before being brought back to the laboratory. The collected leaves were laid flat on a tray lined with water-soaked gauze and surface-sterilized with 75% alcohol. A 1 mL syringe was used to prick both sides of the main vein of the leaf, and 10 μL of bacterial suspension was pipetted onto the prick wound for inoculation. After inoculation, the leaves were placed in an artificial climate chamber at 28 ℃, 75% relative humidity, and a photoperiod of L / D = 12h / 12h. The time of disease onset and the size of lesions were recorded after inoculation. Results showed that the lesion area was smaller and the disease progressed more slowly when the knocked-out bacterial suspension infected detached *Pyrus pyrifolia* leaves compared to normal bacterial suspension infection. Figure 6 ).

[0140] 3. Inoculation trials of Tobacco denudata and Tobacco Benedict.

[0141] Aspirate the bacterial suspension using a 1 mL sterile syringe, remove the needle, and immerse the syringe on the back of the tobacco plant, marking the infiltrated area with a circle. After inoculation, place *Nicotiana macrophylla* and *Nicotiana benthamiana* in an artificial climate chamber at 28 ℃, 75% relative humidity, and a photoperiod of L / D = 12h / 12h. Record the onset time and degree of cell necrosis after inoculation. The results showed that in *Nicotiana macrophylla* and *Nicotiana benthamiana*, the lesion area after knockout was smaller and the onset of disease was slower compared to the normal bacterial suspension. Figure 7 ).

[0142] 4. Experiment on the competitive effects of WT bacterial suspension and ΔHrpG bacterial suspension on young Korla fragrant pears

[0143] Healthy young Korla pears, 3-4 cm in diameter, were harvested and sterilized by soaking in 75% alcohol for 10 minutes. Using a 10 μL sterilization pipette tip, holes approximately 1 cm in diameter were poked into the surface of the young pears. First, 5 μL of WT bacterial solution was inoculated into the poked holes, followed by 5 μL each of WT and ΔHrpG bacterial solutions. The inoculated young pears were then placed flat on trays lined with absorbent cotton, sealed with plastic wrap, and placed in an artificial climate chamber with a photoperiod of L / D = 12 h / 12 ​​h, a temperature of 28 ℃, and a relative humidity of 75%. The area of ​​lesion spread was recorded over a 96-hour period. The results showed that young pears inoculated with WT followed by ΔHrpG bacterial solution exhibited significantly lower pathogenicity, smaller lesion area, and slower disease progression compared to those inoculated solely with WT solution. Figure 8This indicates that by applying the mutant ΔHrpG to plants or their growing environment, biocontrol of fire blight can be achieved through the competitive interaction between the mutant ΔHrpG and the wild-type pathogen.

[0144] sequence list

[0145] The HrpG gene (SEQ ID NO:1), a pear fire blight effector, is derived from Erwinia amylovora from Xinjiang.

[0146] ATGAGATCAACTGAATTGCAGCAGTGGGCGCAGCGCTGGCTCGACGATGTTGGCACAGACCAACAGCTGACGGTGGATGACGGCGTCGTCTGGTTGCAGCGTCGGGCGGATCGGTTTTTTGCGTTGGCAGAGCTGACGATCAATGCTTCGCTGGACGATGAGCTGCTGGGGCGGGCGCTACAGCTTTCGGCCCCGACCCTACGTTATTTTGGCCGC GATGCCGCCGCACTGGCGCAACAAGGCAACAGCCTGATATTAGCGTTAGCTATCCGTCAACTGGAGGTTAACGCCGTCTGTCAACAGCTGGAATCACTGCTTAATCAACGTGATGTCTGGCAGACAATGCTACAACAACCGCGAAGAAAAGCGGCAACCCATGGCGCGGTGCCTTTGCACAGCCTGGCATTTTTACCAAGGGGAAACCATGGTTGA

[0147] HrpG protein (SEQ ID NO:2), a pear blight effector:

[0148] MRSTELQQWAQRWLDDVGTDQQLTVDDGVVWLQRRADRFFALAELTINASLDDELLGRALQLSAPTLRYFGRDAAALAQQGNSLILALAIRQLEVNAVCQQLESLLNQRDVWQTMLQQPRRKAATHGAVPLHSLAFLPRGNHG

[0149] Gentamicin (GM) gene (SEQ ID NO:3):

[0150] ggaaacggatgaaggcacgaacccagttgacataagcctgttcggttcgtaaactgtaatgcaagtagcgtatgcgctcacgcaactggtccagaaccttgaccgaacgcagcggtggtaacggcgcagtggcggttttcatggcttgttatgactgtttttttgtacagtctatgcctcgggcatccaagcagcaagcgcgttacgccgtgggtcgatgtttgatgttatggagcagcaacgatgttacgcagcagcaacgatgttacgcagcagggcagtcgccctaaaacaaagttaggtggctcaagtatgggcatcattcgcacatgtaggctcggccctgaccaagtcaaatccatgcgggctgctcttgatcttttcggtcgtgagttcggagacgtagccacctactcccaacatcagccggactccgattacctcgggaacttgctccgtagtaagacattcatcgcgcttgctgccttcgaccaagaagcggttgttggcgctctcgcggcttacgttctgcccaggtttgagcagccgcgtagtgagatctatatctatgatctcgcagtctccggcgagcaccggaggcagggcattgccaccgcgctcatcaatctcctcaagcatgaggccaacgcgcttggtgcttatgtgatctacgtgcaagcagattacggtgacgatcccgcagtggctctctatacaaagttgggcatacgggaagaagtgatgcactttgatatcgacccaagtaccgccacctaacaattcgt

[0151] Upstream homologous arm gene (LF) of effector HrpG (SEQ ID NO:4):

[0152] GCTGGCAGCAGCAATATCAGCAGCTGGAAGCGCAGGTACTTGAAGTGATGGACAGCGTATTGACGCAGGCGCTTGACCAGTTGTTGACTGACGTTCCGCAAACACAGCGGCTGGCGGCGATATTACGTCAACTGCTACGGGCAAAAACCCTGACTGAACAGGGCAGCCTTTATTGTCATCCGGCACAGCATCTTGAGATCGCCGACTGGTTGCGCAGTCATGACCACCTGGCCTGGCAGCTACAGCCGGATGAGTCGCTGGCGCAAGATAGCCTGAAACTGGTCACCGCTAATGGCGAATTGTCCCTTGACTGGCAGCAGGCAGTGCGCCAGTTACTCCCTCCTCAAACAGCCAGCTAAACCGCTACAAAAAACTTCACTTCACCACGGAACTCCGCCACGCCCGAACCCCACTCAAAGACAGGACCCACTCAATGAGAGAGTTTAATTATGTCATCAATCAGCGCGCTCCAGCGCCGCCTGGACAGTCAGTTTGATAAAGCACAAACCCAACTGGACGATGCCACCTTGGATGCCAGTGAAGGCTACAGCCAGGAGGACAGTTTTGCCTTCTTTGAAGCCAGTATGGGACTTTCCAACGCCAGCTGGGCTGCGAGCCAGGAGCTGACGGTTAAACACGGGCTTGCCAAAGCCATCATCA

[0153] Downstream homologous arm gene (LF) of effector HrpG (SEQ ID NO:5):

[0154] GAAACGAGAGTTGCGCTGCCGGTTGTTGGGCGCGCTATTAATGCTGTGCGCAACACTCCCGGCCGGGGCGCAGACCCCGGCCGACTGGAAAGAACAGTCATACGCCTATTCTGCCGATCGCACGCCGTTATCCACGGTGCTGCAAGATTTTGCTGACGGGCACAGTGTTGATCTTCACCTTGGCAATGTGGAAGACACAGAGGTTACCGCGAAGATCCGCGCTGAAAACGCCAGTGCGTTCCTCGACCGCCTGGCGCTGGAGCACCATTTCCAGTGGTTCGTTTACAACAATACGCTGTACGTCAGCCCTCAGGACGAGCAAAGCTCGGAACGTCTGGAGATCTCCCCGGACGCCGCACCGGATATTAAGCAGGCGCTAAGCGGAATTGGCCTGCTGGACCCGCGTTTTGGCTGGGGAGAACTGCCGGATGATGGCGTGGTGCTGGTGACCGGGCCGCCGCAATATCTGGAGCTGGTTAAGCGCTTCAGTGAGCAACGCGAGAAGAAAGAGGATCGGCGTAAAGTCATGACCTTCCCGCTGCGCTATGCGTCGGTGGCCGACCGCACCATTCATTACCGTGACCAGACGGTGGTGATCCCCGGCGTTGCCACCATGCTGAATGAGCTGATGAACGGTAAACGCGCGGCGCCAGCCAGCGC。

Claims

1. Use of an effector HrpG gene with a nucleotide sequence as set forth in SEQ ID NO: 1 for at least one of the following (a1)-(a2): (a1) preventing and controlling plant fire blight; (a2) preparing a product for preventing and controlling plant fire blight; wherein the plant is pear or tobacco.

2. Use of biological material related to an effector HrpG gene with a nucleotide sequence as set forth in SEQ ID NO: 1 for at least one of the following (a1)-(a2): (a1) preventing and controlling plant fire blight; (a2) preparing a product for preventing and controlling plant fire blight; wherein the biological material related to the effector HrpG gene is a protein encoded by the effector HrpG gene or a biological material for inhibiting or knocking out the effector HrpG gene; wherein the biological material for inhibiting or knocking out the effector HrpG gene is at least one of the following (b1)-(b3): (b1) a nucleic acid molecule for inhibiting or knocking out the effector HrpG gene; (b2) a recombinant vector containing the nucleic acid molecule of (b1); (b3) a recombinant microorganism containing the nucleic acid molecule of (b1) or a recombinant microorganism containing the recombinant vector of (b2); wherein the plant is pear or tobacco. The protein encoded by the effector HrpG gene is a protein as set forth in the following (c1) or (c2): (c1) a protein having an amino acid sequence as set forth in SEQ ID NO: 2; (c2) a fusion protein having the same function obtained by connecting a protein tag to the N-terminus and / or C-terminus of the protein of (c1). The use is at least one of the following (d1)-(d2): (d1) weakening the pathogenicity of a fire blight pathogen by inhibiting or knocking out the effector HrpG gene; (d2) constructing a fire blight pathogen mutant ΔHrpG with a knocked-out effector HrpG gene, and applying the mutant ΔHrpG to a plant or a plant growth environment, so as to achieve biological control of plant fire blight through the competition between the mutant ΔHrpG and a wild-type pathogen; wherein the fire blight pathogen is Erwinia amylovora. The effector HrpG gene is inhibited or knocked out by targeting the effector HrpG gene or a protein encoded by the effector HrpG gene using a gene editing technology, an RNA interference technology or a chemical inhibitor. The method is at least one of the following (d1)-(d2): (d1) weakening the pathogenicity of a fire blight pathogen by inhibiting or knocking out an effector HrpG gene with a nucleotide sequence as set forth in SEQ ID NO: 1; (d2) constructing a fire blight pathogen mutant ΔHrpG with a knocked-out effector HrpG gene with a nucleotide sequence as set forth in SEQ ID NO: 1, and applying the mutant ΔHrpG to a plant or a plant growth environment, so as to achieve biological control of fire blight through the competition between the mutant ΔHrpG and a wild-type pathogen; wherein the fire blight pathogen is Erwinia amylovora; and the plant is pear or tobacco. The fire blight pathogen mutant ΔHrpG is constructed by the following steps:

3. Use according to claim 2, characterized in that, ​ ​ ​ 4. Use according to claim 1 or 2, characterized in that, ​ ​ ​ ​ 5. Use according to claim 4, characterized in that, ​ 6. A method of controlling fire blight, characterized by, ​ ​ ​ ​ ​ 7. A mutant of the fire blight pathogen, Erwinia amylovora, with an effector HrpG gene knockout, ΔHrpG, characterized by: ​ (1) an upstream homologous arm sequence as shown in SEQ ID NO: 4, a downstream homologous arm sequence as shown in SEQ ID NO: 5, and a gentamicin resistance gene sequence as shown in SEQ ID NO: 3 of the amplification effect factor HrpG gene; (2) linking the upstream homologous arm sequence, the gentamicin resistance gene sequence, and the downstream homologous arm sequence to form an LF-GM-RF fusion fragment; (3) cloning the LF-GM-RF fusion fragment into a suicide plasmid pK18mobsacB to construct a recombinant plasmid; (4) introducing the recombinant plasmid into the fire blight pathogen by conjugation transfer, and screening the fire blight pathogen mutant ΔHrpG with the effect factor HrpG gene knocked out through twice homologous recombination; The fire blight pathogen is Erwinia amylovora.

8. A biocontrol agent for Canker disease, characterized by, The bacterial agent comprises the fire blight pathogen mutant ΔHrpG with the effect factor HrpG gene knocked out according to claim 7. The fire blight pathogen is Erwinia amylovora. The bacterial agent comprises the fire blight pathogen mutant ΔHrpG with the effect factor HrpG gene knocked out according to claim 7.

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