Application of magnaporthe oryzae Pmk1 protein K347 site as target for inhibiting pathogenicity of magnaporthe oryzae and preventing and controlling rice blast

Abstract

The invention belongs to the technical field of gene engineering, and provides application of a magnaporthe oryzae Pmk1 protein K347 site as a target for inhibiting the pathogenicity of magnaporthe oryzae. Researches find that after the K347 site of the magnaporthe oryzae Pmk1 protein is mutated, the pathogenicity of magnaporthe oryzae is remarkably reduced, growth and expansion of magnaporthe oryzae hyphae can be inhibited, autophagy in the conidium germination process can be promoted, and secretion of effect protein of magnaporthe oryzae can be inhibited. Compared with the drug design aiming at the whole Pmk1 gene, the drug design aiming at the K347 site disclosed by the invention is accurate in range and has very high specificity and biological safety. The invention provides a new target site for developing a pyricularia grisea antibacterial drug, and the site has good application prospect and value in the aspect of preventing and controlling the rice blast.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and more specifically, relates to the application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for inhibiting the pathogenicity of the rice blast fungus and preventing and controlling the rice blast disease. Background Art

[0002] Rice blast, caused by rice blast fungus, is the number one disease of rice and poses a serious threat to global food production. Prevention and control of rice blast is urgent. The formation of appressorium, penetration of appressorium into the host, and expansion of mycelium are important steps in the infection of rice blast fungus. Understanding the molecular mechanisms of these processes provides an important theoretical basis for the development of specific and efficient fungicides, and can also provide new targets for the design of new fungicides.

[0003] The new nucleic acid pesticide is called the third revolution in the history of pesticides. Nucleic acid pesticides use RNAi technology to target the essential genes of harmful organisms and achieve efficient gene silencing, thereby effectively controlling the occurrence of pests and diseases. Compared with traditional chemical pesticides, nucleic acid pesticides have the advantages of high targeting, easy degradation, rich targets and flexible design. It has been reported that they have good control effects on some plant pathogenic fungi. For this type of technology, the selection of target genes is crucial. The more precise the target gene site, the higher the safety to non-target organisms, especially nucleic acid pesticides that select target gene sites for point mutations, which have a high safety factor.

[0004] The role of MAPK signaling pathway in the process of plant pathogenic fungi infecting hosts and the host's resistance to pathogenic fungi has always been a hot topic of research at home and abroad. Existing technologies show that the Pmk1 gene (a MAPK protein kinase) in rice blast fungi is essential for the formation of appressorium. After the gene is deleted, the rice blast fungi cannot form appressorium, resulting in a complete loss of pathogenicity. However, the signaling pathway MAPK where the Pmk1 gene is located is conserved in all eukaryotic organisms. Developing rice blast fungi nucleic acid pesticides targeting the entire Pmk1 gene will be effective against mammals, beneficial organisms and plants, affecting the growth of crops and human safety. Therefore, Pmk1 is not suitable as a target for drug design due to its conservatism.

[0005] Therefore, finding the highly specific pathogenic sites of rice blast fungus is of great significance for the development of rice blast nucleic acid pesticides with high biosafety. Summary of the invention

[0006] In order to solve the above-mentioned existing problems, the present invention has found through research that after the K347 site of the Pmk1 protein of the rice blast fungus is mutated, the pathogenicity of the rice blast fungus is significantly reduced, and the growth and expansion of the rice blast fungus hyphae are inhibited, the autophagy during the germination of conidia is promoted, and the secretion of the effector protein of the rice blast fungus is inhibited. Therefore, the present invention provides the application of the K347 site of the Pmk1 protein as a target for plant disease control drugs.

[0007] The first object of the present invention is to provide application of K347 site of Pmk1 protein of rice blast fungus.

[0008] The second object of the present invention is to provide the use of a reagent for mutating the K347 site of the Pmk1 protein of the rice blast fungus.

[0009] The third object of the present invention is to provide a nucleic acid pesticide for inhibiting the pathogenicity of rice blast fungus or preventing and controlling plant diseases caused by rice blast fungus.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention found that after the K347 site of the Pmk1 protein of the rice blast fungus was mutated, the pathogenicity of the rice blast fungus was significantly reduced, and the growth and expansion of the rice blast fungus hyphae were inhibited, the autophagy process during conidia germination was promoted, and the secretion of effector proteins of the rice blast fungus was inhibited.

[0012] Therefore, the present invention provides the following application scheme:

[0013] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for inhibiting the pathogenicity of the rice blast fungus.

[0014] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for controlling plant diseases caused by the rice blast fungus.

[0015] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for controlling rice blast.

[0016] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products that inhibit the pathogenicity of the rice blast fungus.

[0017] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for preventing and controlling plant diseases caused by the rice blast fungus.

[0018] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for preventing and controlling rice blast.

[0019] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in inhibiting the mycelial growth of the rice blast fungus.

[0020] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for inhibiting the mycelial growth of the rice blast fungus.

[0021] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in regulating autophagy of conidial cells of the rice blast fungus.

[0022] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products that regulate the autophagy of the conidial cells of the rice blast fungus.

[0023] Application of the K347 site of the rice blast fungus Pmk1 protein as a target in regulating the exocytosis of rice blast fungus effector proteins.

[0024] Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for regulating the exocytosis of effector proteins of the rice blast fungus.

[0025] Specifically, the amino acid sequence of the Pmk1 protein is shown in SEQ ID NO.1, and the K347 site is the 347th position in the amino acid sequence of the Pmk1 protein.

[0026] Optionally, the nucleotide sequence of the Pmk1 gene encoding the amino acid sequence is shown in SEQ ID NO.2, and the K347 site is positions 1039 to 1041 of the nucleotide sequence of the Pmk1 gene.

[0027] At the same time, the present invention also provides the following application scheme:

[0028] Application of a reagent for mutating the K347 site of the Pmk1 protein of the rice blast fungus in the preparation of a product for inhibiting the pathogenicity of the rice blast fungus.

[0029] Application of a reagent for mutating the K347 site of the Pmk1 protein of the rice blast fungus in the preparation of products for preventing and controlling plant diseases caused by the rice blast fungus.

[0030] Application of a reagent for mutating the K347 site of the Pmk1 protein of rice blast fungus in the preparation of products for preventing and controlling rice blast.

[0031] A nucleic acid pesticide for inhibiting the pathogenicity of rice blast fungus or preventing and controlling plant diseases caused by rice blast fungus, comprising an agent capable of mutating the K347 site of the Pmk1 protein of rice blast fungus.

[0032] As an optional embodiment, the reagent capable of mutating the K347 site of the Pmk1 protein of the rice blast fungus, such as the pKN-Pmk1 of the present invention K347R carrier.

[0033] The present invention has the following beneficial effects:

[0034] The present invention has found that after the mutation of the K347 site of the Pmk1 protein of the rice blast fungus, the pathogenicity of the rice blast fungus is significantly reduced, and the growth and expansion of the mycelium of the rice blast fungus are inhibited, the autophagy process during the germination of conidia is promoted, and the secretion of the effector protein of the rice blast fungus is inhibited. The above experimental results prove that the K347 site of the Pmk1 protein of the rice blast fungus is a key site for the pathogenicity of the rice blast fungus. Compared with drug design for the entire Pmk1 gene, the drug designed for the K347 site of the present invention has a precise range and high specificity and biosafety.

[0035] The present invention provides a new target site for developing antibacterial drugs for rice blast fungus, and the site has good application prospects and value in preventing and controlling rice blast disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 For Pmk1 and Pmk1 K347R Molecular docking model with Mst7 protein.

[0037] Figure 2 Schematic diagram of the Pmk1 mutation site.

[0038] Figure 3 Results of pathogenicity determination of different rice blast fungus mutants.

[0039] Figure 4 Figure 2 shows the appressorium formation of different rice blast fungus mutants (Figure A shows the appressorium formation of different rice blast fungus mutants observed under a microscope; Figure B shows the appressorium formation rate of different rice blast fungus mutants).

[0040] Figure 5 The figure shows the infection of barley leaves by different rice blast fungus mutants.

[0041] Figure 6 Laser scanning confocal microscopy was used to observe the results of ATG8-GFP autophagosomes in hyphae and conidia.

[0042] Figure 7 Laser scanning confocal microscopy was used to observe the secretion of the effector protein AVR-Pia through the BIC structure in the infected hyphae.

[0043] Figure 8 ΔPMK1 / PMK1 K343R Results of pathogenicity determination of rice blast fungus mutants. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] Tomato oatmeal medium (OTA medium): boil water, weigh 40g oatmeal, add to boiling water and boil for 30min, filter to extract juice; add 150mL freshly squeezed tomato juice to oatmeal juice, add water to 1L, 1.6% Agar; sterilize with high temperature and high pressure. For the medium for spore production of rice blast fungus, add 0.6g CaCO3 (dissolved in tomatoes first) to 1L medium.

[0047] Complete medium (1L): 6g yeast extract, 3g enzymatically hydrolyzed casein, 3g acid hydrolyzed casein, 10g sucrose, sterilized by high temperature and high pressure.

[0048] LR medium (1 L): Yeast extract (Yeast extract) 1 g, acid hydrolyzed casein (Enzymatic casein hydrolysate) 1 g, sucrose (Sucrose) 342 g.

[0049] SR medium (1 L): Yeast extract (Yeast extract) 1 g, acid hydrolyzed casein (Enzymatic casein hydrolysate) 1 g, sucrose (Sucrose) 342 g, agar (agar) 15 g.

[0050] 2×CTAB solution: 10 mL 1.0 M Tris-HCl (pH 8.0), 40 mL 0.5 M EDTA (pH 8.0), 20 g CTAB and 81.8 g NaCl are dissolved in 800 mL ddH2O, the volume is adjusted to 1 L, and dissolved at 37°C overnight; sterilize under high temperature and high pressure, and store at room temperature.

[0051] 70% ethanol: Mix anhydrous ethanol and ddH2O in a volume ratio of 7:3.

[0052] 0.7M NaCl solution (1L): 40.908g NaCl; sterilize under high temperature and high pressure.

[0053] STC solution (1 L): 1.2 M sorbitol, 10 mM Tris-HCl (pH 7.5), 50 mM CaCl2; sterilized by high temperature and high pressure.

[0054] PTC solution (1 L): 60% polyethylene glycol 3350, 10 mM Tris-HCl (pH 7.5), 50 mM CaCl2; sterilized by high temperature and high pressure.

[0055] Driselase permeate: 20 mg / mL Driselase, prepared with 0.7 M NaCl solution, sterilized by filtration with a 0.22 μm filter.

[0056] 0.025% Tween-20 solution (Tween-20): 250 μL Tween-20, add water to make up to 1L.

[0057] Phenol-form mixture: Tris-saturated phenol, chloroform and isoamyl alcohol are mixed in a volume ratio of 25:24:1.

[0058] The amino acid sequence of Pmk1 is shown in SEQ ID NO.1, SEQ ID NO.1:

[0059] MSRANPPSNSSGSRKISFNVSEQYDIQDVVGEGAYGVVCSAIHKPSGQKVAIKKITPFDHSMFCLRTLREMKLLRYFNHENIISILDIQKPRSYETFNEVYLIQELMETDMHRVIRTQDLSDDHCQYFIYQTLRALKAMHSANVLHRDLKPSNLLLNANCDLKVCDFGLARSAASQED NSGFMTEYVATRWYRAPEIMLTFKEYTKAIDVWSVGCILAEMLSGKPLFPGKDYHHQLTLILDVLGTPTMEDYYGIKSRRAREYIRSLPFKKKVPFRTLFPKTSDLALDLLEKLLAFNPVKRITVEEALKHPYLEPYHDPDDEPTAPPIPEEFFDFDKHKDNLSKEQLKQFIYQEIMR.

[0060] The nucleotide sequence of the Pmk1 gene is shown in SEQ ID NO.2, SEQ ID NO.2:

[0061]

[0062] The sequence of the complementary vector pKN is shown in SEQ ID NO.3, SEQ ID NO.3:

[0063]

[0064] The sequences of the primers in the following examples are shown in Table 1.

[0065] Table 1 Primer sequence list

[0066]

[0067]

[0068] After extensive research on the function and sequence of Pmk1 protein, our team found a point mutation site that can accurately affect the pathogenicity of rice blast fungus, namely the K347 site of Pmk1. After mutating this site, the pathogenicity of rice blast fungus can be significantly and accurately reduced without affecting other indicators such as the formation of appressorium. The following examples show relevant experiments and data.

[0069] Example 1 Pmk1 and Pmk1 K347R Molecular docking models of their interacting protein Mst7

[0070] 1. Experimental methods

[0071] The function of the K347 site of Pmk1 was predicted based on the protein structure. K347R The amino acid sequence of the protein was extracted, and the Mst7 protein was selected from the reported Pmk1 interacting proteins, and its amino acid sequence was extracted. The extracted sequence was imported into alphafold3, and the amino acid sequences of Pmk1 and Mst7, Pmk1 and K347R Molecular structure simulation was performed with Mst7.

[0072] (II) Experimental results

[0073] Pmk1 and Pmk1 K347R Molecular docking model with Mst7 protein Figure 1 The results showed that Pmk1 and Mst7 interacted with each other, and Pmk1 K347R More forces appear in the interaction with Mst7, making the interaction between the two more stable.

[0074] Example 2 Construction of Rice Blast Fungus Δpmk1 / PMK1 K347R and Δpmk1 / PMK1 strains

[0075] The Pmk1 gene number is MGG_09565. Its protein contains a large kinase domain. K347 is located at the C-terminus of the protein. The codon encoding K347 is AAG. K347R -F / Pmk1 K347R-R, mutated the codon of K347 to AGG in the pKN-Pmk1 vector to construct pKN-Pmk1 K347R Vector, schematic diagram of Pmk1 mutation site as shown Figure 2 shown.

[0076] The Pmk1 gene of the rice blast fungus was knocked out by homologous recombination principle to obtain a Δpmk1 knockout mutant (Δpmk1 strain). The specific method is as follows: the 1.5kb sequence upstream of the Pmk1 gene was fused with the hygromycin phosphotransferase gene fragment sequence by fusion PCR method, which was recorded as LB-HY fragment. Similarly, the 1.5kb sequence downstream of the Pmk1 gene was fused with the hygromycin phosphotransferase gene fragment sequence, which was recorded as RB-YG fragment. According to the principle of homologous recombination, the PEG-mediated protoplast transformation method was used to simultaneously transfer the LB-HY fragment and the RB-YG fragment into the protoplasts of the wild-type strain of the rice blast fungus. These two DNA fragments can constitute a complete hygromycin (HYG) fragment and replace the target gene. The HYG resistance was used for screening, and the Δpmk1 strain was obtained by sequencing identification.

[0077] The constructed vectors pKN-Pmk1 and pKN-Pmk1 were transformed and identified according to the following scheme. K347R Transformed into Δpmk1 knockout mutant and identified to obtain Δpmk1 / PMK1 K347R and Δpmk1 / PMK1 strains.

[0078] ΔPMK1 / PMK1 K347R The K347 codon of the Pmk1 gene in the strain was mutated to AGG.

[0079] The K347 codon of the Pmk1 gene in the Δpmk1 / PMK1 strain is the original AAG.

[0080] The specific construction method is as follows:

[0081] 1. Conversion Plan

[0082] (1) Preparation of rice blast fungus protoplasts:

[0083] Inoculate an appropriate amount of rice blast fungus hyphae in 200 mL of complete culture medium, shake and cultivate at 28°C and 150 rpm for 36-48 hours, and collect the mycelium by filtering with three layers of sterile lens paper. After washing the mycelium with precooled 0.7M NaCl solution, transfer it to a sterilized 50 mL centrifuge tube. Add about 1 mL of the crash enzyme permeate for every 1 g of mycelium, and perform enzymolysis at 28°C and 150 rpm for 3-4 hours. Wash the mycelium with 4°C precooled 0.7M NaCl solution, filter through three layers of sterile lens paper, collect protoplasts, centrifuge at 4000 rpm and 4°C for 15 minutes, remove the supernatant, wash the protoplasts with 5 mL of STC solution, and then adjust the protoplast concentration to 0.5-1×10 8 / mL (10-20 protoplasts per small grid of the hemocytometer).

[0084] (2) Construction of pKN-Pmk1 vector and pKN-Pmk1 K347R Carrier

[0085] Construction of pKN-Pmk1 vector (gene complementation vector): Using the genomic DNA of the wild-type strain of rice blast fungus as a template, the Pmk1 gene (including the 1.5kb promoter region of the Pmk1 gene, the gene itself and the 500bp terminator region) was amplified with pKN-pmk1-F / R primers, and the fragment was connected to the complementary vector pKN according to the homologous recombination method. The pKN-Pmk1 vector was verified by sequencing.

[0086] Construction of pKN-Pmk1 K347R Vector: Using the constructed pKN-Pmk1 vector as a template, first use pKN-F / Pmk1 K347R -R primer pair for PCR amplification, and at the same time, pKN-Pmk1 vector was used as a template with Pmk1 K347R -F / pKN-R primer pair for PCR amplification to complete the first round of PCR amplification. Then use pKN-pmk1-F / R primers to perform the second round of PCR amplification with the two products of the first round of PCR as templates. The second round of PCR fragments were connected to the complementary vector pKN according to the homologous recombination method, and sequencing was used to verify pKN-Pmk1 K347R carrier.

[0087] (3) Transformation vector:

[0088] The adjusted concentration of rice blast fungus protoplasts was divided into 50 mL centrifuge tubes, 150 μL per tube, and an equal volume of the constructed pKN-Pmk1 vector or pKN-Pmk1 was added. K347RVector (1.5-2.0μg), add STC solution to 300μL, and place on ice for 20min. Then slowly add PTC solution (2mL / tube) drop by drop, and let it stand on ice for 20min. Add pre-cooled STC solution (25mL / tube), slowly mix, and centrifuge at 4000rpm and 4℃ for 15min. Remove the supernatant, then add 3mL of LR culture medium to each tube, and culture at 28℃ for about 12-13h.

[0089] (4) Resistance screening:

[0090] Pour the transformed LR liquid culture medium into a sterilized 9 cm culture dish, add SR culture medium cooled to about 50°C, mix well, wait for it to solidify, and then spread a layer of water agar culture medium (containing 250 μg / mL Geneticin G418) cooled to about 50°C on top, culture at 28°C for 4-6 days, and transfer the resulting transformants to OTA culture medium for culture.

[0091] 2. Transformant Identification Scheme

[0092] After the transformants were cultured for 3-5 days, a small amount of genomic DNA was extracted. The specific steps were as follows:

[0093] 1. Use a toothpick to scrape the mycelium into a 2 mL centrifuge tube (EP), add magnetic beads to the centrifuge tube, place it on a cell disruptor and grind it for 20 seconds, then add 650 μL of 2×CTAB extract, mix well, heat at 65°C for 30 minutes, and invert and mix 2-3 times during the process.

[0094] 2. Then transfer all the liquid to a new centrifuge tube, let it cool to room temperature, add an equal volume of phenol-mimetic mixture, invert and mix thoroughly, and centrifuge at 12000 rpm at room temperature for 15 minutes.

[0095] 3. After absorbing the supernatant, add 0.6 times the volume of isopropanol, place in a -20°C refrigerator for precipitation for 30 minutes, and centrifuge at 12000 rpm, 4°C for 15 minutes.

[0096] 4. Discard the supernatant, rinse the precipitate twice with 70% ethanol and once with anhydrous ethanol, dry it naturally or in a vacuum, and dissolve it in 30 μL sterile water to obtain transformant DNA.

[0097] The transformant DNA was used as a template and a pair of detection primers Pmk1CK-F / Pmk1CK-R were used for PCR amplification. The reaction system and procedure are shown in Table 2.

[0098] Table 2 Reaction system and procedure

[0099]

[0100] The successfully amplified PCR product was sequenced, and the sequencing primer Pmk1CK-F was selected. The sequencing result was compared with the original sequence. If AAG at K347 changed to AGG, it was considered that the midpoint mutation of the strain was successful.

[0101] After identification, Δpmk1 / PMK1 was constructed as above. K347R strain and Δpmk1 / PMK1 strain.

[0102] Example 3 Pmk1 K347 site affects the determination of pathogenicity of rice blast fungus

[0103] 1. Experimental methods

[0104] Method for obtaining conidia of rice blast fungus: inoculate the rice blast fungus strain on OTA culture medium, place it in an inverted culture in a 28℃ light incubator, grow for 5 days, use a red-hot bacteria ring to fully interrupt the mycelium in a sterile operating table, and then evenly spread it on a new OTA plate, place it in an inverted culture in a 28℃ light incubator for 36 hours, wash off the newly grown mycelium with a cotton swab, cover it with a double layer of gauze, and place it in a 28℃ light incubator for 48 hours. Conidia can be obtained after 48 hours.

[0105] According to the method for obtaining conidia of rice blast fungus in this example, wild-type strain (WT), Δpmk1 / PMK1 K347R Conidia of strain and Δpmk1 / PMK1 strain.

[0106] The method for detecting the pathogenicity of the strain is:

[0107] S1: Wash the conidia on the OTA plate with 0.025% Tween-20 solution, filter into a 50 mL centrifuge tube using a funnel made of three layers of lens paper to obtain a conidia suspension, measure the conidia concentration of the conidia suspension under a microscope using a hemocytometer, and adjust the conidia concentration to 3 × 10 4 Pieces / mL;

[0108] S2: Spray the adjusted conidia suspension evenly on the surface of barley leaves cultured to the one-leaf-one-heart stage with a nozzle for inoculation (the nozzle is tilted 45° during inoculation to ensure that the conidia liquid lands on the leaf surface), spray Tween-20 solution on the inner wall of the plastic bottle, cover the entire pot of barley leaves, and culture with a black cloth;

[0109] S3: Expose to light after 24 hours, continue to keep moist for 48 hours, then unscrew the cap of the plastic bottle; 5 days after inoculation, take photos to record the disease status of the barley leaves.

[0110] According to the pathogenicity detection method of this example, the wild-type strain (WT), Δpmk1 / PMK1 K347RThe pathogenicity of the strain and the Δpmk1 / PMK1 strain was determined.

[0111] (II) Experimental results

[0112] The results of pathogenicity determination of different rice blast fungus mutants are shown in Figure 3 The results showed that the barley leaves inoculated with the wild-type (WT) strain and the Δpmk1 / PMK1 strain were seriously diseased, with a large number of yellow spots appearing on the inoculated leaves, indicating that the WT strain and the Δpmk1 / PMK1 strain were highly pathogenic. K347R The barley leaves of the strain only showed individual lesions, indicating that Δpmk1 / PMK1 K347R The pathogenicity of the strain was significantly reduced.

[0113] Example 4 Pmk1 K347 site does not affect the formation of rice blast appressorium

[0114] 1. Experimental methods

[0115] According to the method for obtaining conidia of rice blast fungus in Example 3, WT strain, Δpmk1 strain, Δpmk1 / PMK1 strain, and K347R Conidia of strain and Δpmk1 / PMK1 strain.

[0116] Wash the conidia with sterile water to obtain a conidia suspension. Use a funnel made of three layers of lens paper to filter the conidia suspension into a 2 mL centrifuge tube. Use a hemocytometer to measure the spore concentration of the conidia suspension under a microscope. Adjust the conidia concentration to 2 × 10 5 / mL, then spot the conidia suspension on a square hydrophobic glass slide in 4 rows at a volume of 3 μL per drop, with 4-5 drops in each row, place it in a culture dish to keep it moist, culture it in the dark for 24 hours, and observe and count the formation of appressorium under a microscope.

[0117] (II) Experimental results

[0118] Appressorium formation of different rice blast fungus mutants Figure 4 The results showed that the Δpmk1 strain (complete knockout of Pmk1 protein) did not form appressorium at all, while the WT strain and Δpmk1 / PMK1 K347R strains and Δpmk1 / PMK1 strains can form appressoria normally, with appressoria formation rates all above 80%, and there is no significant difference in the appressoria formation rates among the three strains, indicating that the K347 site of the Pmk1 protein does not affect the appressoria formation of rice blast fungus.

[0119] Example 5 Pmk1 K347 site inhibits the infection process of rice blast fungus

[0120] 1. Experimental methods

[0121] According to the method for obtaining conidia of rice blast fungus in Example 3, WT strain, Δpmk1 strain, Δpmk1 / PMK1 strain, K347R Conidia of strain and Δpmk1 / PMK1 strain.

[0122] The conidia were washed with 0.025% Tween-20 solution to obtain a conidia suspension, which was then filtered through a funnel made of three layers of lens paper into a 2 mL centrifuge tube. The conidia concentration of the conidia suspension was measured under a microscope using a hemocytometer. The conidia concentration was adjusted to 1 × 10 5 Pieces / mL.

[0123] Place the first leaf of barley that has grown to the one-leaf-one-heart stage with the back side facing up in a petri dish covered with absorbent paper (already moistened), and press down the two ends with moistened absorbent cotton. Apply 3 μL of the adjusted conidia suspension to a row on the left and right sides of the veins on the back of the barley leaf, 5-7 drops per row, and then culture in the dark at 28°C to keep moist. Take the lower epidermis of the barley 24h and 36h after inoculation and observe under a microscope.

[0124] (II) Experimental results

[0125] The infection of different rice blast fungus mutants on barley leaves Figure 5 As shown, the results show that:

[0126] At 24 hpi, most of the WT and Δpmk1 / PMK1 strains formed infectious hyphae, and nearly half of the infectious hyphae had branched to form secondary infectious hyphae, while the Δpmk1 / PMK1 K347R Most strains remained in the appressorium stage, and only a few appressorium formed primary infectious hyphae, indicating that Δpmk1 / PMK1 K347R The strain is defective in appressorium penetration.

[0127] At 36 hpi, the infectious hyphae in the WT strain and the Δpmk1 / PMK1 strain had filled the entire host cell, and many of the infectious hyphae successfully penetrated the host cell into the adjacent host cell, while the Δpmk1 / PMK1 K347R The infective hyphae formed branches normally and filled the infected host cells, but most of the infective hyphae did not spread to the surrounding host cells and appeared unable to expand.

[0128] In summary, the above results indicate that the K347 mutation of the Pmk1 protein leads to defects in the invasion of appressoria and the spread of infectious hyphae to surrounding host cells, thereby reducing its pathogenicity.

[0129] Example 6 Pmk1 K347 site negatively regulates the autophagy process of rice blast fungus

[0130] 1. Experimental methods

[0131] According to the transformation scheme of Example 2 (the difference is that the transformation vector is different), the ATG8-GFP vector was transferred into the WT strain and Δpmk1 / PMK1 K347R Among the strains, the successfully transformed fluorescent transformants WT / ATG8-GFP strain and Δpmk1 / PMK1 were screened by fluorescence microscopy. K347R +ATG8-GFP strain.

[0132] According to the method for obtaining conidia of rice blast fungus in Example 3, WT / ATG8-GFP strain and Δpmk1 / PMK1 were obtained. K347R Conidia of +ATG8-GFP strain.

[0133] Wash the WT / ATG8-GFP strain and Δpmk1 / PMK1 with sterile water. K347R The conidia of the +ATG8-GFP strain were placed under a laser scanning confocal microscope (Confocal) to observe the number and signal intensity of ATG8 autophagosomes in the conidia.

[0134] Inoculate appropriate amount of WT / ATG8-GFP strain and Δpmk1 / PMK1 in 200 mL complete medium. K347R The hyphae of the +ATG8-GFP strain were cultured at 28°C for 36 h, and then the vegetative hyphae were placed under a laser scanning confocal microscope (Confocal) to observe the number and signal intensity of ATG8 autophagosomes in the vegetative hyphae.

[0135] (II) Experimental results

[0136] Laser scanning confocal microscopy was used to observe the ATG8-GFP autophagosomes in hyphae and conidia. Figure 6 As shown, the results showed that ΔPMK1 / PMK1 K347R The number of Atg8 autophagosomes in the conidial vegetative hyphae of the strain was significantly higher than that of the wild-type strain, indicating that the K347 site of the Pmk1 protein negatively regulates the autophagy process of the rice blast fungus. Acceleration or inhibition of the autophagy process during conidia germination will affect the function of the appressorium. K347R The increase in autophagosomes in the strain will lead to excessive cell autophagy, affecting the nutrient supply during the formation of appressorium, and ultimately leading to Δpmk1 / PMK1 K347R The strain was able to form appressorium, but the appressorium function was defective, resulting in decreased pathogenicity.

[0137] Example 7 Pmk1 K347 site regulates the exocytosis of effector proteins in infected hyphae

[0138] 1. Experimental methods

[0139] According to the transformation scheme of Example 2 (the difference is that the transformation vector is different), the AVR-Pia-GFP vector was transferred into the WT strain and Δpmk1 / PMK1 K347R strains, and successfully transformed fluorescent transformants WT / AVR-Pia-GFP strain and Δpmk1 / PMK1 were screened by fluorescence microscopy. K347R +AVR-Pia-GFP strain.

[0140] According to the method for obtaining conidia of Magnaporthe oryzae in Example 3, WT / AVR-Pia-GFP strain and Δpmk1 / PMK1 were obtained. K347R Conidia of +AVR-Pia-GFP strain.

[0141] Wash WT / AVR-Pia-GFP and Δpmk1 / PMK1 with sterile water. K347R +AVR-Pia-GFP strain to obtain conidia suspension, filter the conidia suspension into a 2 mL centrifuge tube using a funnel made of three layers of lens paper, and measure the conidia concentration of the conidia suspension under a microscope using a hemocytometer. Adjust the conidia concentration to 2 × 10 5 Pieces / mL.

[0142] Place rice leaf sheaths that have grown for about 2 months in a culture dish to keep them moist, with the base of the leaf sheath facing downward. Inject the adjusted conidia suspension into the rice leaf sheath with a syringe until the entire leaf sheath is filled with the conidia suspension, and then culture at 28°C in the dark for 24 hours to keep them moist.

[0143] After 24 hours, the surface cells of the leaf sheath were cut off with a blade and placed under a laser scanning confocal microscope (Confocal) to observe the secretion of the effector protein AVR-Pia in the infected hyphae through the biotrophic interfacial complex (BIC) structure.

[0144] (II) Experimental results

[0145] Laser scanning confocal microscopy observations Figure 7 The results showed that AVR-Pia-GFP was clearly localized to the BIC structure in the WT strain and secreted into the host cell by the rice blast fungus through the BIC structure, while in the Δpmk1 / PMK1 K347RNo obvious BIC structure was observed, and the multiple fluorescence signals were diffusely distributed in the cytoplasm, indicating that the K347 site of the Pmk1 protein regulates the exocytosis of effector proteins in the infected hyphae.

[0146] The secretion of effector proteins will affect the expansion of the infective hyphae in the host. The results of this example show that AVR-Pia-GFP (representative of effector proteins) is secreted in Δpmk1 / PMK1 K347R The secretion of the strain was blocked, indicating that the mutation of the Pmk1K347 site would affect the exocytosis of the effector protein in the infectious hyphae, thereby affecting the expansion of the infectious hyphae and leading to a decrease in pathogenicity.

[0147] Comparative Example 1: Mutation of Pmk1 K343 site to R does not affect the pathogenicity of rice blast fungus

[0148] 1. Experimental methods

[0149] Design of mutation primer Pmk1 K343R -F / R, using mutation primers to mutate the codon K343 on the pKN-Pmk1 vector to AGG, constructing pKN-Pmk1 K343R carrier.

[0150] According to the transformation scheme of Example 2 (the difference is that the transformation vector is different), pKN-Pmk1 K343R The vector was transferred into the Δpmk1 knockout mutant to obtain Δpmk1 / PMK1 K343R strains.

[0151] According to the strain virulence detection method of Example 3, WT strain, Δpmk1 / PMK1 K343R The pathogenicity of the strain and the Δpmk1 / PMK1 strain was determined.

[0152] (II) Experimental results

[0153] ΔPMK1 / PMK1 K343R The results of pathogenicity determination of rice blast fungus mutants are as follows Figure 8 As shown, the results showed that after the K343 site mutation of Pmk1, its pathogenicity had no significant difference from that of the wild type and Δpmk1 / PMK1 complement strains, indicating that the K343 site did not affect the pathogenicity of rice blast fungus, and the effect of the K347 site on the pathogenicity of rice blast fungus was specific.

[0154] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for inhibiting the pathogenicity of the rice blast fungus.

2. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for preventing and controlling plant diseases caused by the rice blast fungus.

3. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target for controlling rice blast.

4. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products that inhibit the pathogenicity of the rice blast fungus.

5. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for preventing and controlling plant diseases caused by the rice blast fungus.

6. Application of the K347 site of the Pmk1 protein of the rice blast fungus as a target in the preparation of products for the prevention and control of rice blast.

7. Application of reagents for mutating the K347 site of Pmk1 protein of rice blast fungus in the preparation of products for inhibiting the pathogenicity of rice blast fungus.

8. Use of an agent for mutating the K347 site of the Pmk1 protein of the rice blast fungus in the preparation of a product for preventing and controlling plant diseases caused by the rice blast fungus.

9. Use of a reagent for mutating the K347 site of the Pmk1 protein of the rice blast fungus in the preparation of products for preventing and controlling rice blast.

10. A nucleic acid pesticide for inhibiting the pathogenicity of rice blast fungus or preventing and controlling plant diseases caused by rice blast fungus, characterized in that: Contains reagents capable of mutating the K347 site of the Pmk1 protein of the rice blast fungus.

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