A polypeptide ligand targeting membrane proteins of pathogenic oomycetes, SR-22, and uses thereof

Abstract

The application discloses a polypeptide ligand SR-22 targeting a membrane protein of a pathogenic oomycete and an application thereof, and the amino acid sequence of the polypeptide ligand SR-22 is SRITRLLRGSGSGSSRITRLLR. The polypeptide ligand SR-22 provided by the application can significantly inhibit the growth of various crop pathogenic oomycetes, and can well inhibit the infection of pathogenic bacteria and prevent the occurrence of diseases in practical application, and can be developed into a novel targeted polypeptide pesticide for green prevention and control of crop oomycete diseases.

Description

Technical Field

[0001] This invention relates to a polypeptide ligand SR-22 that targets a conserved key membrane protein of pathogenic oomycetes and its application, belonging to the field of crop disease control in the discipline of plant protection in agronomy. Background Technology

[0002] Oomycetes belong to the phylum Oomycota and class Oomycetes in the kingdom Chromista. They are diploid eukaryotic microorganisms that resemble fungi (haploid) but are more closely related to diatoms and brown algae. The class Oomycetes currently comprises 15 orders, 24 families, nearly 100 genera, and approximately 1500 species. These numerous species are mostly plant pathogens with wide host ranges and high pathogenicity, harming important crops such as grains, vegetables, flowers, and fruit trees. For example, pathogenic Phytophthora species such as Phytophthora muscarinica, Phytophthora soybeani, Phytophthora capsici, and Phytophthora camphorata can cause blight and fruit drop in potatoes, soybeans, melons, vegetables, and trees, resulting in trillions of dollars in crop yield losses worldwide each year due to crop blight. Pythium species such as Pythium tumefaciens, Pythium cerevisiae, and Pythium spp. attack crop roots, causing crop death. Peronophythora species such as Peronophythora lychee downy mildew damages lychee through airborne transmission, causing flower and fruit rot. These oomycete pathogens all cause significant losses to agricultural production. Currently, with the rapid development of modern agriculture and the transformation and upgrading of planting structures in my country, oomycete diseases pose a major challenge to agricultural production and the ecosystem.

[0003] Oomycetes can spread through various pathways, including soil, rainwater, and airflow, and are characterized by short incubation periods, rapid spread, strong infectivity, and multi-site damage. After invading the host, the pathogen quickly forms numerous zoosporangia and releases zoospores, creating a source of reinfection and causing rapid disease spread, making control extremely difficult. Currently, both chemical and agricultural control methods for oomycete diseases are ineffective. This is due to several factors: the unique transmission and infection characteristics of oomycetes; the limited variety of fungicides available; and the rapid mutation and high genetic diversity of oomycetes in the field, which easily leads to drug resistance and loss of resistance in varietals. Furthermore, the extensive use of single chemical pesticides has resulted in the "3R" problem (reduction of pesticide use, reduction of water use, and reduction of organic matter). These problems hinder the implementation of my country's current pesticide reduction policy and the sustainable development of modern agriculture. Therefore, exploring new, efficient, and green methods for controlling oomycete diseases is of paramount importance.

[0004] Studies have revealed significant differences between oomycetes and fungi in cell wall structure, genetics, physiology, biochemistry, and pathogenicity. Therefore, identifying key proteins in the growth, development, and pathogenicity of oomycetes can serve as novel targets for disease control agents, contributing to the development of new drugs.

[0005] Peptide ligands are short peptides (also known as polypeptides or small peptides) that bind to target proteins with high specificity and high affinity, selected from a randomly synthesized library of amino acids. They achieve specific effects by interfering with and disrupting the function of the target protein. Since their introduction in 1990, peptide ligands have been widely used as targeted drugs in basic clinical research, drug design, and targeted disease therapy in the medical field, and the market prospects for peptide drugs are broad. In recent years, with the development of this technology, the application potential of peptide ligands in crop disease control has gradually gained attention. However, there are currently no peptide-targeted drugs for controlling oomycete diseases in crops available globally, and no peptide-targeted drugs developed in my country have been registered or reported. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a polypeptide ligand SR-22 that targets a conserved key membrane protein of the pathogenic oomycetes and its application. This polypeptide ligand can significantly inhibit the occurrence of oomycete diseases in crops.

[0007] Technical solution: To solve the above technical problems, the present invention provides a polypeptide ligand SR-22 that targets a conserved key membrane protein of pathogenic oomycetes, the amino acid sequence of which is SRITRLLRGSGSGSSRITRLLR.

[0008] The present invention also provides a method for obtaining the polypeptide ligand SR-22, comprising the following steps:

[0009] 1. Screening of conserved proteins in oomycetes

[0010] Protein sequences of seven oomycetes (Phytophthora sojae, Phytophthora ramorum, Phytophthorainfestans, Phytophthora capsici, Pythium ultimum, Pythium aphanidermatum, and Hyalloperonospora arabidopsidis) were downloaded. Bioinformatics methods were used to compare the whole genome sequences of these seven oomycetes with those of plants, animals, fungi, bacteria, viruses, and nematodes. The oomycete-specific and highly conserved protein PcMEM1 was screened out, and its protein domains were predicted and analyzed.

[0011] 2. Functional Analysis of PcMEM1

[0012] The full-length PcMEM1 gene of *Phytophthora capsici* was forward cloned into the vector pGFPN, and then transformed into *Phytophthora capsici* strains via protoplast transformation to obtain PcMEM1 overexpressing transformants. The morphological characteristics and pathogenicity changes of the overexpressing transformants during their growth and development stages (hyphae, zoosporangia, and zoospores) were observed.

[0013] Meanwhile, the PcMEM1 gene was transiently expressed in leaves of Nicotiana benthamiana, and the leaf phenotype was observed.

[0014] 3. Screening and identification of targeted peptides

[0015] Randomly composed oligonucleotide fragments were assembled into the pGADT7-derived plasmid pLIB2, and then amplified in E. coli to obtain a polypeptide ligand library. The gene encoding the membrane protein PcMEM1 was cloned into pGBKT7 as bait, and the polypeptide library was screened by yeast two-hybrid. Plasmid DNA was extracted from positive colonies and sequenced to obtain polypeptide sequences. The polypeptide plasmid and bait protein plasmid were co-transformed into yeast for yeast two-hybrid verification.

[0016] The present invention also provides a reagent or drug containing the polypeptide ligand SR-22.

[0017] The present invention also provides the use of the polypeptide ligand SR-22 or the reagent or drug in inhibiting the growth of oomycetes.

[0018] The present invention also provides the application of the polypeptide ligand SR-22 or the reagent or drug in the control of crop oomycete diseases.

[0019] The oomycetes are those that use the conserved key membrane protein PcMEM1 as a key factor for growth, development, infection, and pathogenicity.

[0020] The oomycetes include fungi of the genera *Phytophthora*, *Peronophythora*, *Phytopythium*, or *Pythium*.

[0021] The oomycetes include *Phytophthora capsici*, *Phytophthorainfestans*, *Phytophthora sojae*, *Phytophthora cactorum*, *Peronophythora litchii*, *Phytopythium helicoides*, *Pythium aphanidermatum*, *Pythium vexans*, or *Pythium ultimum*.

[0022] The effective inhibitory concentration of the polypeptide ligand SR-22 is 0.0003–0.3517 mM.

[0023] The crops mentioned include potatoes, chili peppers, or lychees.

[0024] This invention also provides a plate inhibition test for the polypeptide ligand SR-22: SR-22 was added to rye V8 solid medium or PDA medium to prepare plates containing 200, 300, and 500 μM polypeptides. Nine plant pathogenic oomycetes of different trophic types (Phytophthora capsici, Phytophthorainfestans, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium ultimum, Pythium vexans, and Pythium aphanidermatum) were inoculated, and the inhibitory effect of SR-22 on their growth was determined.

[0025] This invention also provides a foliar spraying experiment: a greenhouse potted potato (Solanum tuberosum) variety Désirée was selected, and a 500 μM SR-22 polypeptide solution was sprayed on the leaves. After 12 hours, the leaves were inoculated with a suspension of zoospores of Phytophthora infestans, and the control effect of SR-22 on potato late blight was determined.

[0026] This invention also provides a root irrigation experiment: the pepper (Capsicum annuum) variety "Hanyu Extra Large Horn Pepper" was selected. A 500 μM polypeptide solution was irrigated to the roots of potted pepper seedlings in a greenhouse. After 8 hours, the roots were inoculated with a suspension of Phytophthora capsicum zoospores, and the effectiveness of SR-22 in controlling root blight in peppers was determined.

[0027] This invention also provides a fruit immersion test: Fruits of the litchi (Litchi chinensis) variety "Guiwei" were selected. The litchi fruits were thoroughly immersed in a 500 μM SR-22 polypeptide solution, and after 2.5 h, they were inoculated with Phytophthora litchii. The effectiveness of SR-22 in controlling litchi downy mildew was then determined.

[0028] This invention also provides peptide antibacterial specificity analysis: Non-target fungus *Magnaporthegrisea* and non-target bacteria *Escherichia coli* DH5α strain were selected. SR-22 was added to their culture media to final concentrations of 200, 300, and 500 μM, respectively, and the fungi or bacteria were inoculated. The colony growth inhibition rate of *Magnaporthegrisea* was calculated, and the absorbance (OD) of *Escherichia coli* was measured every hour.600 The study aimed to analyze whether the peptides had an inhibitory effect on the growth of non-target microorganisms (fungi and bacteria).

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention targets a conserved key membrane protein PcMEM1 involved in the growth, development and pathogenicity of oomycetes, and for the first time develops a polypeptide ligand SR-22 that targets and chelates it using biotechnology; 2. The polypeptide ligand SR-22 significantly inhibits the growth of oomycetes, but does not inhibit the growth of non-target fungi and bacteria, and has good specificity; 3. The polypeptide ligand SR-22 has good antibacterial and anti-disease effects, and is expected to be used as a new, safe and environmentally friendly polypeptide pesticide for the green control of oomycete diseases in crops. Attached Figure Description

[0030] Figure 1 Conservation analysis of PcMEM1 in oomycetes;

[0031] Figure 2 Domain analysis of the PcMEM1 protein;

[0032] Figure 3 Subcellular localization of PcMEM1 in overexpressing transformant hyphae; where A, hyphae under a laser confocal microscope; B, PcMEM1 protein expression detected by Western blot.

[0033] Figure 4 To investigate the effects of PcMEM1 overexpression on the growth, development, and pathogenicity of Phytophthora capsici; the study included: A) observation of growth and development characteristics of different strains; B) comparison of growth rates of different strains; C) comparison of zoosporangium numbers of different strains (under 10x objective); D) comparison of zoospore concentrations of different strains; E) comparison of dormant spore germination rates of different strains; F) comparison of pathogenicity of different strains; and G) comparison of lesion area caused by different strains.

[0034] Figure 5 PcMEM1 and its four oomycete homologs induced cell death in tobacco leaf samples; A, expression of PcMEM1 and its four oomycete homologs 4 days after Agrobacterium injection, with GFP as a negative control; B, detection of protein expression by Western blot.

[0035] Figure 6 To obtain the peptide SR-22 using yeast two-hybrid screening; wherein, A, the growth of yeast in yeast strain AH109 containing bait protein PcMEM1 on a deficiency medium lacking tryptophan (W), leucine (L), adenine (A), and histidine (H); B, the growth of yeast colonies on the above four deficiency medium containing x-α-gal for 2 days.

[0036] Figure 7 To verify the interaction between peptide SR-22 and bait protein PcMEM1 (i.e. Pc127259);

[0037] Figure 8 The synthesized SR-22 physical specimen;

[0038] Figure 9 Mass spectrometry data for peptide SR-22;

[0039] Figure 10 High performance liquid chromatography data for peptide SR-22;

[0040] Figure 11 Different concentrations of the polypeptide SR-22 inhibited the growth of nine oomycetes;

[0041] Figure 12 To investigate the use of SR-22 foliar spray to prevent potato late blight, the study included: A) the disease incidence on potato leaves after spraying with 500 μM SR-22 solution and control ddH2O, followed by inoculation with Phytophthora pathogens 3 days later; B) a quantitative comparison of leaf lesion area after spraying with 500 μM SR-22 solution and control ddH2O, followed by inoculation with Phytophthora pathogens 3 days later; and C) a comparison of Phytophthora pathogen biomass in potato leaves after spraying with 500 μM SR-22 solution and control ddH2O, followed by inoculation with Phytophthora pathogens 6 days later (asterisks *** indicate significant differences, P < 0.001).

[0042] Figure 13 To investigate the role of SR-22 in preventing root blight in chili peppers; A, the disease incidence in chili peppers 3 days after irrigating the roots with 500 μM peptide SR-22 solution and control ddH2O followed by inoculation with Phytophthora capsici zoospores; B, statistical graphs of lesion length on chili pepper roots 4 days after inoculation with Phytophthora capsici following ddH2O and SR-22 treatments (* indicates significant difference, P<0.01).

[0043] Figure 14 To prevent the occurrence of downy mildew in litchi by soaking the fruit with SR-22; A, the disease incidence of litchi fruit after soaking the surface of litchi fruit three times with 500 μM SR-22 solution and control ddH2O and then inoculating with Phytophthora litchiensis for 68 h; B, statistical chart of disease severity data of litchi fruit after inoculation with Phytophthora litchiensis for 68 h after treatment with SR-22 and ddH2O. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0045] Example 1: Screening of conserved proteins in oomycetes

[0046] 1. Download all protein sequences encoded by the genomes of seven oomycetes (Phytophthora sojae, Phytophthora ramorum, Phytophthora infestans, Phytophthora capsici, Pythium ultimum, Pythium aphanidermatum, and Hyalloperonosporaarabidopsidis) from the JGI database (https: / / genome.jgi.doe.gov / portal);

[0047] 2. All protein sequences of the above 7 oomycetes were self-aligned using blastP (E-value < 1e-5) using seqhunter software, and then clustered using MCL (Markov Cluster Algorithm) software using TribeMCL software to group homologous sequences into the same orthogroup. Then, only those orthogroups containing homologous sequences of all 7 oomycetes were selected.

[0048] 3. Highly conserved orthogroups proteins in oomycetes were compared with proteins encoded by the genomes of 19 plants, 13 animals, 32 fungi, 17 bacteria, 5 viruses, and 8 nematodes in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) using blastP (E-value < 1e-5). Orthogroups that were dissimilar to those in non-oomycete species were selected as final candidate orthogroups. Based on this, the oomycete-specific and highly conserved protein PcMEM1 (protein ID Pc127259 in the JGI Phytophthora capsici database, NCBI Protected Undisclosed Accession Number PQ277303) was screened out. The evolutionary clustering relationship is as follows: Figure 1As shown: PcMEM1, Pc008439, DVH05_005936, and DVH05_024782 are from *Phytophthora capsici*, PITG22036 and PITG08183 are from *Phytophthora in festans*, PAG1G010589 is from *Pythium aphanidermatum*, KAK1930365.1 is from *Phytophthora citrophthora*, KAG7379021.1 is from *Phytophthora pseudosyringae*, KUG01621.1 is from *Phytophthoranicotianae*, KAG2764292.1 is from *Phytophthora cactorum*, KAH7482102.1 is from *Phytophthora amorum*, and KAE8899104.1 is from *Phytophthora*. fragariae, POM76769.1 from Phytophthorapalmivora, CAH0493029.1 from Peronospora farinosa, CAI5703558.1 from Peronospora effusa, RLN94750.1 from Nothophytophthora sp., KAI9916111.1 from Peronosclerosporasorghi, CAI5741223.1 from Hyloperonospora brassicae, TDH72102.1 from Bremialactucae, KAF1329390.1 from Globisporangium splendens, XP_024582811.1 from Plasmopara halstedii; large red dots indicate proteins that can induce cell death in tobacco leaves;

[0049] Table 1. Other species whose genomes were compared with oomycetes.

[0050]

[0051]

[0052]

[0053]

[0054] 4. The protein domains of PcMEM1 were predicted using SMART software, and the results were as follows ( Figure 2The data shows that PcMEM1 contains 5 transmembrane domains, where TM is the transmembrane domain and ICD (intracellular domain) is the intracellular portion, with numbers indicating amino acid positions.

[0055] Example 2: PcMEM1 gene function analysis

[0056] The full-length PcMEM1 gene of *Phytophthora capsici* (sequence ID 127259 in the JGI *Phytophthora capsici* database, https: / / mycocosm.jgi.doe.gov / Phyca11 / Phyca11.home.html, NCBI Protected Undisclosed Entrance Number PQ277303) was positively cloned into the universal vector pGFPN (enzyme digestion system: 0.5 μL each of PacI and NheI, 5 μL of 10× Buffer, 1 μg DNA, and sterile water to a final volume of 50 μL. The enzyme digestion reaction was incubated at 37°C for 3 h. Ligation conditions: 0.1 μL T4 DNA Ligase, 0.15 pmol of gene fragment, 0.015 pmol of linearized vector, and 10× T4 DNA Ligase Buffer). 1 μL, add sterile water to make up to 10 μL. The ligation reaction was incubated at 16℃ for 12 h. The ligation reaction was then carried out by fusion of the C-terminus of the Ham34 gene (NCBI accession number X16984) of *Phytophthora capsici* with GFP protein via CaCl2-PEG mediated protoplast transformation (for specific methods, refer to the literature published by Chen Xiaoren et al. Chen XR, Zhang Y, Li HY, Zhang ZH, Sheng GL, Li YP, Xing YP, Huang SX, Tao H, Kuan T, Zhai Y, Ma W. The RXLR Effector PcAvh1 Is Required for Full Virulence of *Phytophthora capsici*. Mol Plant Microbe). Interact. 2019, 32(8): 986-1000. doi: 10.1094 / MPMI-09-18-0251-R.) was transformed into protoplasts of *Phytophthora capsici* strain Pc537. G418-resistant transformants were obtained through protoplast regeneration and selection with 37.5 μg / mL G418. GFP fluorescence of the transformants was observed under a fluorescence microscope, revealing positive transformants OE1, OE2, and OE3 expressing green fluorescence. Western blot analysis was used to detect the expression level of PcMEM1 in the overexpressing transformants, confirming them as PcMEM1 overexpressing transformants. The OE2 transformant showed the highest expression level of this protein. The hyphal fluorescence of these *Phytophthora capsici* PcMEM1 gene overexpressing transformants was observed, and the results ( Figure 3 This indicates that the protein is an oomycete membrane protein. Among them, Figure 3WT represents the wild-type strain; Ctrl represents the transformation control; OE1, OE2, and OE3 are all PcMEM1 overexpressing transformants; the excitation wavelength is 488 nm, bar = 10 μm; the antibody is GFP antibody; the morphological characteristics of the gene overexpression transformants at different growth and development stages (hyphae, zoosporangia, zoospores) were observed under a microscope, and their infectivity and pathogenicity were observed after inoculation with Nicotiana benthamiana. Figure 4 42 hours after inoculation, gene overexpression significantly weakened the pathogenicity of *Phytophthora capsici*, and 87 hours after inoculation, it severely affected the growth and development of *Phytophthora capsici*. The growth rate of all three overexpressing transformants was significantly reduced, with increased mycelial branching, twisting, and swelling, higher zoosporangium aberration rate, significantly smaller zoosporangium size, and significantly reduced zoospore number and germination rate. Pathogenicity on *Tobacco Benedict* leaves was significantly decreased. These results indicate that *PcMEM1* is a key factor in the growth, development, and pathogenicity of oomycetes. Attempts to knock out this gene in *Phytophthora capsici* using CRISPR / Cas9 technology failed to obtain transformants, further demonstrating that this protein is a conserved key protein in oomycetes, and knocking out this gene leads to strain death. Figure 4 Figure A shows the colony characteristics (first row; photographed 87 h after inoculation, bar = 1 cm) of wild-type (WT), transformation control (Ctrl), and PcMEM1 overexpression transformants (OE1, OE2, OE3), as well as hyphal morphology (second row; bar = 50 μm), zoosporangium production (third row; bar = 100 μm), and resting spore germination (fourth row; bar = 50 μm). In Figure F, the inoculation amount of zoospores for WT, Ctrl, OE1, and OE2 was adjusted to 1500, and the inoculation amount for OE3 was 20.

[0057] Simultaneously, the genes PcMEM1, Pc008439, PITG22036, PITG08183, and PAG1_G010589 were cloned into the universal plasmid pBin. The restriction enzyme digestion system for PcMEM1, Pc008439, PITG22036, and PITG08183 was: 0.5 μL each of BamHI and KpnI, 5 μL of 10×Buffer, 1 μg of DNA, and sterile water to a final volume of 50 μL. The digestion reactions were incubated sequentially at 30℃ and 37℃ for 2 h each. The restriction enzyme digestion system for PAG1_G010589 was: 0.5 μL each of SalI and KpnI, 5 μL of 10×Buffer, 1 μg of DNA, and sterile water to a final volume of 50 μL. The digestion reaction was incubated at 37℃ for 3 h. Uniform ligation conditions: T4 DNA Ligase. 0.1 μL of gene fragment, 0.15 pmol of linearized vector, 0.015 pmol of 10×T4 DNA Ligase Buffer, and sterile water to a final volume of 10 μL were added. The ligation reaction was incubated at 16°C for 12 h. The mixture was then transformed into Agrobacterium GV3101 strain and added to the OD240 LC50 solution. 600 When the protein expression level was 0.6, the mixture was incubated with 10 mmol / L 4-morpholine ethanesulfonic acid, 0.1 mmol / L acetylsalicylic acid, and 10 mmol / L MgCl2 for 3 h, and then injected into the leaves of *Nicotiana benthamiana*. The plants were cultured in a greenhouse at 20-22℃, and the leaf phenotype was observed visually. Western blot was used to detect protein expression. Results ( Figure 5 The results showed that PcMEM1 and its homologous proteins induced cell death in tobacco leaves, while the expression of the proteins was normal.

[0058] Example 3: Screening and Identification of Targeted Peptides

[0059] 1. Utilize BamHI and ApaI restriction sites (restriction system: 0.5 μL each of BamHI and ApaI, 5 μL of 10× buffer, 1 μg DNA, and sterile water to a final volume of 50 μL. Incubate the digestion reaction sequentially at 30℃ and 37℃ for 2 h each. Ligation conditions: 0.1 μL T4 DNA Ligase, 0.15 pmol fragment, 0.015 pmol linearization vector, and 10× T4 DNA Ligase Buffer). 1 μL of sterile water was added to bring the volume to 10 μL. The ligation reaction was incubated at 16°C for 12 h. The oligonucleotide fragment (5'-GGCAGAGTGGATCCAA(NNK)8GGCTCCGGTTCTGGCTCT(NNK)8AAGGGGCCCCTT-3') was inserted into the pGADT7-derived universal plasmid pLIB2 (AD vector). The plasmid was then amplified in *E. coli* DH5α cells, and 1 × 10⁻⁶ cells were collected in LB medium containing 50 μg / mL ampicillin. 6The plasmid was purified by SDS alkaline lysis to obtain a polypeptide ligand library.

[0060] 2. The gene encoding the membrane protein PcMEM1 was cloned into the universal plasmid pGBKT7 (BD vector for short);

[0061] 3. Using PcMEM1 as the bait protein, peptide ligands capable of specifically targeting and binding to the bait protein PcMEM1 were screened from a peptide ligand library using yeast two-hybrid technology. Figure 6 ); Among them, the sequence of the polypeptide SR-22 was obtained by isolating plasmids and sequencing them from the blue positive colonies shown in the box;

[0062] 4. Plasmid DNA was extracted from positive colonies grown on yeast four-deficient medium (-LWHA) and sequenced to obtain the specific sequence of polypeptide SR-22.

[0063] 5. The obtained polypeptide plasmid SR-22 and bait protein plasmid PcMEM1 were co-transformed into yeast for yeast two-hybrid verification to confirm that SR-22 can interact with the target protein PcMEM1. Figure 7 The text on the left indicates different combinations of yeast two-hybrids. BD is an abbreviation for vector pGBKT7, AD is an abbreviation for vector pLIB2, and the RecT-AD+Lam-BD combination is a positive control. The top shows the four-cell culture medium used. 3-AT is 3-amino-1,2,4-triazole, a competitive inhibitor of yeast HIS3 protein (His3p). In the inset, each group of four colonies is a 10-fold serial dilution from left to right, and the colonies were photographed after 4 days of growth on the medium. SR-22 was synthesized by Shanghai Jietai Biotechnology Co., Ltd. Figure 8 , Figure 9 , Figure 10 The amino acid sequence of the obtained polypeptide SR-22 is SRITRLLRGSGSGSSRITRLLR.

[0064] Example 4 Plate Antibacterial Test

[0065] 1. Inoculate 6mm diameter Phytophthora infestans mycelial blocks onto rye V8 solid medium and incubate in the dark at 18℃ for 14 days before use. Inoculate Phytophthora capsici, Phytophthora sojae, Phytophthora cactorum, Peronophythora litchii, Phytopythium helicoides, Pythium ultimum, Pythium vexans, and Pythium aphanidermatum onto PDA medium and incubate in the dark at 25℃ for 7 days before use.

[0066] 2. Dissolve SR-22 polypeptide powder in sterile water to prepare a 50mM stock solution. Add 28, 42, and 70 μL of the polypeptide stock solution to PDA (suitable for *Phytophthora capsici*, *Phytophthora sacchariformis*, *Phytophthora hymexazol*, *Phytophthora lychee*, *Pythium scutellarioides*, *Pythium cerevisiae*, *Pythium scutellarioides*, *Pythium scutellarioides*, *Pythium scutellarioides*, and *Pythium scutellarioides*) or rye V8 medium (suitable for pathogenic *Phytophthora*) that has been melted and cooled to about 50°C. After thorough mixing, pour the solution into sterile petri dishes (60 mm in diameter). Add 7 mL of medium to each petri dish. Finally, prepare drug-containing plates with polypeptide concentrations of 200, 300, and 500 μM. Meanwhile, the medium plates without polypeptides serve as a negative control group.

[0067] 3. Using a sterile punch, take a 6mm diameter fungal cake from the edge of the freshly cultured colony. Using a sterile inoculation needle, inoculate a 6mm diameter fungal cake into the center of the drug-containing plate (mycelial side down). Seal the petri dish with sealing film and invert the pathogenic Phytophthora plate at 18℃. Invert other oomycete plates in a constant temperature incubator at 25℃ and incubate in the dark.

[0068] 4. Observe and photograph the colony growth within 1–7 days after inoculation. Measure the colony diameter using the cross-sectional method and calculate the colony growth inhibition rate. The colony growth inhibition rate is calculated using the following formula: Colony growth inhibition rate = [(Control colony diameter - Treated colony diameter) / Control colony diameter] × 100%. Based on the antibacterial test results, calculate the virulence equation of SR-22 against oomycetes and the effective inhibitory concentration using probability analysis.

[0069] Depend on Figure 11It can be seen that polypeptide SR-22 has a good inhibitory effect on *Phytophthora capsici*, *Phytophthora causalina*, *Phytophthora soybeani*, *Phytophthora hymexazol*, *Phytophthora litchiensis*, *Pythium styracifolium ... and *Pythium styracifolium*. These nine pathogens come from four different genera of the Oomycetes class, namely *Phytophthora*, *Phytophthora*, *Pythium styracifolium*, and *Pythium*. It can be seen that polypeptide SR-22 has a broad-spectrum inhibitory effect on the growth of Oomycetes.

[0070] SR-22 significantly inhibited the mycelial growth of nine oomycetes, and its inhibitory effect was dose-dependent. At a concentration of 500 μM, SR-22 inhibited the colony growth of *Phytophthora capsici*, *Phytophthora virosa*, *Phytophthora sacchariformis*, *Phytophthora hymexazol*, *Phytophthora lychee*, *Pythium styracifolium*, *Pythium citrinum*, *Pythium styracifolium*, and *Pythium terrestris* by 84.47%, 78.58%, 86.00%, 69.67%, 92.40%, 78.95%, 70.65%, 79.17%, and 82.61%, respectively, indicating that this polypeptide has a highly significant antibacterial effect on oomycetes. Figure 11 The text on the left side of the image shows the Latin names of the tested oomycetes, and the concentration of SR-22 used is shown above. The images show Phytophthora capsici (2 days after inoculation), Phytophthora infestans (7 days after inoculation), Phytophthora sojae (84 hours after inoculation), Phytophthora cactorum (2 days after inoculation), Peronophythora litchii (78 hours after inoculation), Phytopythium helicoides (20 hours after inoculation), Pythium aphanidermatum (2 days after inoculation), Pythium vexans (1 day after inoculation), and Pythium ultimum (20 hours after inoculation).

[0071] Based on the results of the antibacterial test, the virulence equation of SR-22 against oomycetes and the effective inhibitory concentration were calculated using probability analysis. The results are shown in Table 2.

[0072] Table 2. Antibacterial activity of peptide SR-22 against 9 species of oomycetes.

[0073]

[0074] Example 5: Plant Leaf Spraying Experiment

[0075] 1. Select the potato (Solanum tuberosum) variety Désirée, one of the staple crops. Transplant the sprouted potato tubers into plastic pots containing sterile soil and cultivate them in a greenhouse with alternating light and dark conditions at 22-25℃ for 16h / 8h for 4-6 weeks before use.

[0076] 2. Culture the pathogenic fungus Phytophthora in the above manner for 14 days, rinse the culture plate with sterile distilled water, and scrape the surface of the plate with a sterile spreader to collect sporangia. Then place the collected sporangia suspension in a 4℃ refrigerator for 15-30 min and a 18℃ incubator for 20-30 min in sequence to stimulate the sporangia to release zoospores.

[0077] 3. Prepare SR-22 polypeptide stock solution (50mM). Use a pipette to take 30μL and dilute it with sterile water to 3mL to prepare 500μM polypeptide working solution for later use.

[0078] 4. Cut potato leaves of uniform size, rinse the leaves with sterile water to remove impurities, and carefully wipe off any remaining water. Turn the leaves over and spray 3 mL of polypeptide working solution evenly on the back of the leaves, repeating the spraying 2-3 times. The control group was sprayed with the same amount of sterile water. Each group had 4 leaves treated. Place the leaves in a 25℃ greenhouse for about 12 hours to dry them. Then, inoculate the back of the leaves with 10 μL of zoospore suspension (concentration of 60 spores / μL). After inoculation, place the leaves in a sterile plastic box lined with moistened filter paper, seal the plastic box with plastic wrap, and place the box in an 18℃ incubator. Observe the pathogen infection results and take photos for 2-6 days after inoculation.

[0079] 5. The biomass of Phytophthora pathogenica in leaves was measured by qPCR (reaction system and procedure are shown in Tables 3 and 4, primer sequences are shown in Table 5). Six days after inoculation, total genomic DNA was extracted from an area of ​​equal size from the inoculation site on the leaves. Using the potato housekeeping gene StEF1α (NCBI accession number NM_001288491) as a reference control, the relative expression level of the Phytophthora pathogenica housekeeping gene Pief2α (NCBI accession number XM_002901697) was analyzed to measure the biomass of Phytophthora pathogenica per unit area of ​​leaf.

[0080] Table 3. qPCR reaction system (20 μL)

[0081]

[0082] Table 4 qPCR reaction procedures

[0083]

[0084] The primer sequences used (Table 5) are shown below:

[0085] Table 5 Primer sequences

[0086]

[0087]

[0088] 6. After spraying potato leaves with a 500 μM polypeptide SR-22 solution, inoculate with Phytophthora infestans for 3 days and observe the disease development. Measure the relative biomass of Phytophthora infestans in the leaves at 6 days to evaluate the control effect of potato late blight.

[0089] Spraying potato leaves with a 500 μM polypeptide SR-22 solution, followed by inoculation with pathogenic Phytophthora, resulted in the following disease progression after 3 days: Figure 12 As shown in A and 12B, the relative biomass of pathogenic Phytophthora in the leaves at 6 days is as follows: Figure 12 As shown in C. Figure 12 Therefore, SR-22 can effectively inhibit the infection of pathogenic Phytophthora and control the occurrence of potato late blight when sprayed on leaves.

[0090] Example 6: Plant Root Drenching Experiment

[0091] 1. Select the chili pepper (Capsicum annuum) variety "Hanyu Extra Large Horn Pepper", sprout it, and then transplant it into plastic pots containing sterile soil. Place it in a greenhouse with alternating light and dark conditions at 22-25℃ for 16h / 8h, and let it grow for 4-6 weeks before use.

[0092] 2. Pour 15-20 mL of 10% V8 culture medium into sterile petri dishes (9 cm in diameter). Transfer 8-10 pieces of freshly cultured *Phytophthora capsici* mycelium into the culture medium. Incubate in the dark at 25°C for 2-3 days. Once the mycelial colony has formed, discard the culture medium, add 15-20 mL of sterile water to resuspend the mycelial colony, and continue incubation at the same temperature. Change the water every 12-24 hours, for a total of 2-3 times, until a large number of zoosporangia are formed. To obtain zoospores, place the petri dishes sequentially in a 4°C refrigerator for 10-15 minutes and then in a 25°C incubator for 10-30 minutes to stimulate zoospore release.

[0093] 3. Prepare a 50mM SR-22 polypeptide stock solution. Use a pipette to take 500μL and dilute it with sterile water to 50mL to prepare a 500μM polypeptide solution for later use.

[0094] 4. Irrigate the roots of pepper plants with 8 mL of 500 μM polypeptide solution. Plants irrigated with an equal volume of sterile water serve as a control. There are 6 plants in each treatment and control group. After placing the plants in a greenhouse for 8 hours, irrigate the roots of the pepper plants again with 6 mL of a *Phytophthora capsici* zoospore suspension (concentration of 50 zoospores / μL). Observe the disease development of the pepper plants and take photographs for 3-6 days after root irrigation. Results are as follows: Figure 13As shown, compared with the control group, the degree of infection of pepper roots by Phytophthora capsici after SR-22 pretreatment was significantly inhibited, and the occurrence of pepper blight was significantly controlled. It can be seen that the polypeptide SR-22 can be used to inhibit the infection of pathogenic oomycetes in the roots of peppers and even other plants.

[0095] Example 7 Fruit Immersion Test

[0096] 1. Culture Phytophthora lycheeis for 4-6 days before use;

[0097] 2. Choose fresh, healthy lychee of the "Guiwei" variety (Litchi chinensis);

[0098] 3. Prepare SR-22 polypeptide stock solution (50mM). Use a pipette to take 50μL and dilute it with sterile water to 5mL to prepare a 500μM polypeptide working solution for later use.

[0099] 4. Rinse the surface of fresh lychee fruits thoroughly with sterile water, then blot dry with absorbent paper. Immerse the lychee fruits repeatedly in a 500μM SR-22 polypeptide solution, using fruits immersed in an equal amount of sterile water as a control. There are 8 fruits in each group. Allow the fruits to air dry at room temperature for 2.5 hours. Using a 6mm diameter sterile punch, collect mycelial blocks of *Phytophthora lychee* from the edge of the colony. Place the mycelial-containing side firmly against the fruit surface, seal with plastic wrap, and keep in a moisturizing plastic box. Incubate at 25℃ for 2–4 days, observing and photographing the disease development of the lychee fruits during this period. The results are as follows: Figure 14 As shown, compared with the control, treatment of litchi fruit with 500 μM SR-22 polypeptide solution significantly reduced the incidence of litchi downy mildew, indicating that polypeptide SR-22 can inhibit the infection of pathogenic oomycetes on litchi and even other plant fruits through spraying or soaking. The disease severity was divided into four grades: Grade 0 (no lesions), Grade 1 (lesion area <10%), Grade 2 (lesion area 10%–30%), Grade 3 (lesion area 30%–50%), and Grade 4 (lesion area 50%–100%).

[0100] Example 8: Analysis of the antibacterial specificity of the peptide

[0101] 1. Magnaphalthe grisea, the causal agent of rice blast, was inoculated onto PDA agar plates and cultured in the dark at 25°C for 1–7 days. Plates containing 200, 300, and 500 μM peptides were prepared using the previous method, with a peptide-free plate serving as a negative control. A 6 mm diameter mycelial disc was punched from the edge of freshly cultured colonies using a sterile punch. A disc was inoculated into the center of the drug-containing plate (mycelial side down) using a sterile inoculation needle. The plate was sealed with sealing film and incubated upside down in a 25°C incubator in the dark. The colony growth inhibition rate and effective inhibition concentration were calculated using the previous method. The results showed that even at a concentration as high as 500 μM, SR-22 only inhibited the growth of Magnaphalthe grisea by 11.31%, with an EC50 value of 319.869 mg / mL, indicating that SR-22 did not inhibit the growth of Magnaphalthe grisea.

[0102] 2. Culture Escherichia coli DH5α strain overnight (12-13 h) in LB liquid medium at 37℃ and 200 rpm, then add LB medium and allow its OD to rise. 600 Dilute to 0.1, then add peptide solution to achieve final concentrations of 200, 300, and 500 μM, respectively. Continue culturing E. coli, and measure OD every 1 hour. 600 The culture was continued until 5 hours after incubation. Results showed that the addition of SR-22 to the liquid culture medium did not significantly affect the growth of *E. coli*. At 5 hours of incubation, the OD values ​​of bacteria in the control and 500 μM SR-22 treatment groups were significantly lower. 600 The values ​​were 1.6485 and 1.448, respectively, showing no significant difference, indicating that SR-22 does not inhibit the growth of Escherichia coli. In conclusion, SR-22 does not have a significant inhibitory effect on non-target microorganisms (i.e., fungi and bacteria).

Claims

1. A polypeptide ligand SR-22 targeting the membrane protein of pathogenic oomycetes, characterized in that, Its amino acid sequence is SRITRLLRGSGSGSSRITRLLR.

2. A reagent or drug containing the polypeptide ligand SR-22 of claim 1.

3. The application of the polypeptide ligand SR-22 of claim 1 or the reagent or drug of claim 2 in inhibiting the growth of oomycetes, characterized in that, The oomycetes include Phytophthora capsici ( Phytophthora capsici ), pathogenic fungus ( Phytophthora infestans ), soybean phytotoxicum ( Phytophthora sojae ), Phytophthora ( Phytophthora cactorum ), Phytophthora indicum ( Peronophythora litchii ), Pythium stalkii ( Phytopythium helicoides ), Pythium spp. ( Pythium aphanidermatum ), clockwork mold ( Pythium vexans ) or ultimate pyrophyllosis ( Pythium ultimum ).

4. The application of the polypeptide ligand SR-22 of claim 1 or the reagent or drug of claim 2 in the control of crop oomycete diseases, characterized in that, The oomycetes include Phytophthora capsici ( Phytophthora capsici ), pathogenic fungus ( Phytophthora infestans ), soybean phytotoxicum ( Phytophthora sojae ), Phytophthora ( Phytophthora cactorum ), Phytophthora indicum ( Peronophythora litchii ), Pythium stalkii ( Phytopythium helicoides ), Pythium spp. ( Pythium aphanidermatum ), clockwork mold ( Pythium vexans ) or ultimate pyrophyllosis ( Pythium ultimum ).

5. The application according to any one of claims 3 to 4, characterized in that, The effective inhibitory concentration of the polypeptide ligand SR-22 is 0.0003~0.3517 mM.

6. The application according to claim 4, characterized in that, The crops mentioned include potatoes, peppers, or lychees.

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