Gene plirp1 and application thereof in preventing and treating litchi blight

Abstract

The application discloses a gene PlIRP1 and application thereof in prevention and treatment of litchi blight, and belongs to the technical field of green prevention and control of crop diseases.The application applies a protoplast transformation technology based on polyethylene glycol and a CRISPR / Cas9 knockout strategy, constructs a PlIRP1 knockout vector, and uses PEG-mediated protoplast transformation and a homologous recombination method to knockout the gene PlIRP1 from litchi blight Peronophythora litchii, so as to obtain a knockout mutant.Compared with a wild type, the mutant has obviously weakened pathogenicity and oospore production.The application proves that PlIRP1 is necessary for sexual compatibility and infection pathogenicity of the litchi blight Peronophythora litchii.The research is helpful to further clarify the pathogenic molecular mechanism of the litchi blight Peronophythora litchii, and provides a target gene for development of an effective fungicide.

Description

Technical Field

[0001] This invention belongs to the field of green prevention and control technology for crop diseases, specifically involving the gene PlIRP1 and its application in the prevention and control of lychee downy mildew. Background Technology

[0002] Downy mildew, caused by *Peronophythora litchii* Chen ex Ko et al., is the most serious disease affecting litchi production. Due to the lack of effective resistant litchi varieties, current control measures primarily rely on spraying chemical pesticides, which not only pose a high risk of pesticide resistance but also pollute the environment. Therefore, researching the pathogenic mechanism of *Peronophythora litchii* and developing green control technologies is of great significance.

[0003] Studies have shown that successful infection of litchi by Phytophthora licheniformis mainly depends on a series of pathogenic factors. Therefore, identifying key pathogenic genes related to Phytophthora licheniformis and conducting functional studies can provide candidate drug targets for the control of litchi downy mildew and is of great significance for breeding disease-resistant varieties. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of existing technologies for controlling downy mildew on litchi, the present invention aims to provide a pathogenicity-related protein PlIRP1 (Peronophythora litchiiinfection-related protein 1) of downy mildew on litchi.

[0005] This invention discloses a previously unknown protein, PlIRP1, and its encoding gene, PlIRP1, from *Phytophthora lichei*. The full-length PlIRP1 gene is shown in SEQ ID NO.1, has no introns, and encodes 63 amino acids as shown in SEQ ID NO.2. This invention utilizes CRISPR / Cas9 gene editing technology to construct pBSSK::PlIRP1 and PYF2.3G-ribo-sgRNA::PlIRP1 knockout vectors, and then knocks out the PlIRP1 gene using PEG-mediated protoplast transformation. The final knockout mutants T83 and T123 (named according to the transformant verification sequence) exhibit significant defects in the growth and development of *Phytophthora lichei*. Similarly, using CRISPR / Cas9 gene editing technology, in situ complementation vectors pBSSK::NPTII, PYF2.3G-ribo-sgRNA1::NPTII, and PYF2.3G-ribo-sgRNA2::NPTII were constructed, ultimately yielding complemented strain C140. Pathogenicity assays showed that the knockout mutants T83 and T123 of the PlIRP1 gene significantly reduced the pathogenicity of litchi fruit (variety: Huaizhi), and the complemented strains of the PlIRP1 gene restored the pathogenicity of C140 to the level of the wild-type strain. These experiments demonstrate that the PlIRP1 gene of *Phytophthora litchii* is an important pathogenicity-related gene of *Phytophthora litchii*.

[0006] A litchi downy mildew-associated protein, PlIRP1, has the amino acid sequence shown in SEQ ID NO.2, or a similar sequence as shown in SEQ ID NO.2 obtained by substitution, insertion, or deletion of one or more amino acids, still having the same or similar function.

[0007] The aforementioned biological materials related to the PlIRP1 protein, a pathogenic protein associated with Phytophthora downy mildew of litchi, are any one or more combinations of the following biological materials:

[0008] 1) The nucleic acid molecule encoding the pathogenic protein PlIRP1 of Phytophthora downy mildew;

[0009] 2) An expression cassette containing the nucleic acid molecules described in 1);

[0010] 3) A recombinant vector containing the nucleic acid molecules described in 1), or a recombinant vector containing the expression cassette described in 2);

[0011] 4) Recombinant microorganisms containing the nucleic acid molecules described in 1), or recombinant microorganisms containing the expression cassette described in 2), or recombinant microorganisms containing the recombinant vector described in 3);

[0012] 5) Nucleic acid molecules that inhibit or block the gene expression of the litchi downy mildew-associated protein PlIRP1;

[0013] 6) Gene knockout vector prepared using the nucleic acid molecules described in 5) to inhibit or block the gene expression of the litchi downy mildew-associated protein PlIRP1;

[0014] 7) The gene-deficient Phytophthora litchii pathogenicity-related protein PlIRP1 was prepared using the gene knockout vector described in 6).

[0015] Further, the nucleic acid molecule mentioned in 1) is the gene sequence of the litchi downy mildew pathogenicity-related protein PlIRP1, as shown in SEQ ID NO:1, or a similar sequence as shown in SEQ ID NO:1 obtained by base insertion, deletion, or substitution that still has the same or similar function.

[0016] Furthermore, the nucleic acid molecule mentioned in 5) is the antisense RNA, siRNA, shRNA, or sgRNA of the PlIRP1 gene.

[0017] Furthermore, the sgRNA is as follows:

[0018] sgRNA: 5'-CTAGCCAGCGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCGCTGCCAGTTCTTGGTCT-3'.

[0019] The application of the aforementioned litchi downy mildew-associated protein PlIRP1 or biological materials is any one or more combinations of the following applications:

[0020] i) Application in regulating the pathogenicity of downy mildew on litchi;

[0021] ii) Application in regulating the infectivity of downy mildew on litchi;

[0022] iii) Application in regulating the formation of oospores of Phytophthora downyensis on litchi;

[0023] iv) Application in the prevention and control of lychee downy mildew;

[0024] v) Application as a target in the design and screening of drugs against downy mildew of litchi.

[0025] A method for preventing and controlling litchi downy mildew caused by Phytophthora licheeis is achieved by inhibiting or blocking the gene expression of the pathogenic protein PlIRP1 of Phytophthora licheeis.

[0026] A drug screening model for resistance to Phytophthora litchii, wherein the drug screening model is a gene-deficient Phytophthora litchii with the pathogenicity-related protein PlIRP1.

[0027] The method for constructing the above-mentioned drug screening model against Phytophthora licheniformis includes the following steps:

[0028] (1) Based on the PlIRP1 gene sequence, sgRNA was designed using the sgRNA website. The sgRNA was then ligated with the pYF2.3G-Ribo-sgRNA vector to obtain the PlIRP1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlIRP1.

[0029] Alternatively, primers for amplifying the left and right homologous arms can be designed based on the PlIRP1 gene sequence, and the left and right homologous arms can be amplified using Phytophthora litchii genomic DNA as a template; the obtained left and right homologous arms of the PlIRP1 gene and the NPTII gene fragment can be ligated with the pBSSK vector to obtain the PlIRP1 gene knockout plasmid pBSSK::PlIRP1.

[0030] (2) The PlIRP1 gene knockout plasmids pYF2.3G-Ribo-sgRNA::PlIRP1 and pBSSK::PlIRP1 were introduced into the protoplasts of wild-type strains of Phytophthora litchii. After screening and verification, the PlIRP1 gene knockout mutant was obtained, which is the drug screening model of Phytophthora litchii.

[0031] Furthermore, the sgRNA described in step (1) is as follows:

[0032] sgRNA: 5'-CTAGCCAGCGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCGCTGCCAGTTCTTGGTCT-3'.

[0033] Furthermore, the amplification primers for the left and right homologous arms mentioned in step (1) are as follows:

[0034] Left homologous arm amplification primers:

[0035] PlIRP1-Left-F: 5'-AGAACTAGTGGATCCCCCGGGATCACCGACATGGAGGAGCAG-3';

[0036] PlIRP1-Left-R: 5'-ATCTTGTTCAATCATGTCTGTCGGATGTAGGAGACTGTG-3';

[0037] Right homologous arm amplification primers:

[0038] PlIRP1-Right-F: 5'-GACGAGTTCTTCTGAACGCGCAGAAGGAGAGAAGAG-3';

[0039] PlIRP1-Right-R: 5'-ATCGAATTCCTGCAGCCCGGGCTATCAGGAGCAGGAGACAGCGT-3'.

[0040] The present invention has the following advantages and effects compared with the prior art:

[0041] This invention demonstrates through experiments that by amplifying the upstream and downstream sequences of the PlIRP1 gene, and then amplifying the NPTII gene sequence, ligating them into a PBSSK vector, and performing homologous recombination, the gene is knocked out using polyethylene glycol (PEG)-mediated protoplast transformation and a CRISPR / Cas9 knockout strategy. Due to the deletion of the PlIRP1 gene, the resulting mutant exhibits significantly reduced oospore yield and pathogenicity compared to the wild type. This invention confirms that the PlIRP1 gene is essential for the formation and pathogenicity of oospores of *Phytophthora litchifolia*. Our research contributes to elucidating the pathogenic molecular mechanism of *Phytophthora litchifolia* and provides a theoretical basis for discovering novel drug targets and designing novel, highly effective, low-toxicity, and safe fungicides. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the construction of homologous recombination of the PlIRP1 gene from Phytophthora litchii.

[0043] Figure 2 These are agarose gel electrophoresis images of PCR verification of *Phytophthora licheniformis* PlIRP1 gene knockout transformants and complement transformants, as well as quantitative PCR amplification results of *Phytophthora licheniformis* PlIRP1 gene knockout transformants and complement transformants; where a is the electrophoresis image and b is the quantitative PCR amplification result; lane WT (Wild type): wild type of *Phytophthora licheniformis*; lane CK (Control): transformants transformed with PEG into empty vector without knockout of the target gene; lanes T83 and T123 represent two knockout transformants of gene PlIRP1, respectively; lane C140 represents the complement transformant of gene PlIRP1.

[0044] Figure 3 The results of pathogenicity analysis of zoospores from the PlIRP1 knockout mutants T83 and T123 inoculated on litchi fruit are shown in Figure a. Among them, a shows the pathogenicity results of zoospores from WT, CK, PlIRP1 knockout mutants and complement strains inoculated on litchi fruit 4 days after inoculation, and the image is three representatives of each case; b shows the lesion length measured 4 days after zoospore inoculation on fruit.

[0045] Figure 4The results of pathogenicity analysis of litchi fruit inoculated with mycelium cakes from gene PlIRP1 knockout mutants T83 and T123 are shown in Figure 1. Among them, a shows the pathogenic results of litchi fruit 4 days after inoculation with mycelium cakes from WT, CK, PlIRP1 knockout mutants and complement strains, and the image is three representatives of each example; b shows the length of lesions measured 4 days after mycelium cake inoculation on leaves.

[0046] Figure 5 The results show the oospore yield analysis of the PlIRP1 knockout mutants T83 and T123; where A is the oospore morphology diagram with a scale bar of 100 μm, and B is the statistical result of oospore yield. Detailed Implementation

[0047] The present invention will be further described in detail below with reference to the actual situation and the accompanying drawings, but the implementation method of the present invention is not limited thereto.

[0048] Unless otherwise specified, the following implementation plan will generally follow standard testing conditions or the testing conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0049] Example 1: Construction of the PlIRP1 gene knockout vector

[0050] Test materials

[0051] Test strains, plants and vectors

[0052] The tested strain was the wild-type strain of *Peronophythora litchii* (Wild Type, abbreviated WT), a common *Peronophythora* species that can be obtained commercially or naturally. The *Escherichia coli* strain DH5α was also obtained commercially. The inoculated plant material was litchi (Huaizhi) fruit. The oomycete knockout and transformation vectors pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA (disclosed in the literature "Yufeng, Fang, Linkai, et al. Efficient Genome Editing in the Oomycete *Phytophthora sojae* Using CRISPR / Cas9[J]. Current Protocols in Microbiology, 2017"), and pBSSK (disclosed in the literature "ACRISPR / Cas9-mediated in situ complementation method for *Phytophthora sojae* mutants[J]. Molecular Plant") were used. The above vectors were kindly provided by the Laboratory of Oomycetes and Fungi Molecular Biology, College of Plant Protection, Nanjing Agricultural University. (Published in Pathology, 2021, 22(3)).

[0053] Main test culture medium

[0054] Carrot agar (CA) (1L): Juice 300g of carrots, filter through gauze, and sterilize at 121℃ for 20min. Add 1.5% (w / v) agar powder to the solid medium.

[0055] LB medium (1L): 5g yeast extract, 10g tryptone, 10g sodium chloride (NaCl), sterilized at 121℃ for 20min. For solid medium, add 1.5% (w / v) agar powder.

[0056] Nutrition Pea Broth (NPB) (1L): Boil 120g of fresh peas in water for 20 minutes, then filter. Add 5g D-Sorbitol, 5g D-Mannitol, 5g glucose, 3g potassium nitrate (KNO3), 2g calcium carbonate (CaCO3), 2g yeast extract, 1g dipotassium hydrogen phosphate (K2HPO4), 1g potassium dihydrogen phosphate (KH2PO4), 0.5g magnesium sulfate (MgSO4), 0.1g calcium chloride (CaCl2), 2mL vitamin stock, and 2mL trace elements to a final volume of 1L with ddH2O. Sterilize at 121℃ for 20 minutes. For solid culture, add 1.5% (w / v) Difco Bacto Agar.

[0057] Pea mannitol medium (Pea / 0.5mol·L⁻¹) -1 Manitol (PM) (1L): Boil 120g of fresh peas in water for 20 minutes, then filter. Add 91g of D-Mannitol, 2g of CaCO3 and 1.32g of CaCl2 to the filtrate, and bring the volume to 1L with ddH2O. Sterilize at 121℃ for 20 minutes. For solid culture medium, add 1.5% (w / v) Difco Bacto Agar.

[0058] Piper's medium (1L): 0.5g potassium dihydrogen phosphate, 0.25g magnesium sulfate heptahydrate (MgSO4·7H2O), 1g L-asparagine, 1mg vitamin B1, 0.5g yeast extract, 10mg β-sitosterol, and 5g glucose. Add ddH2O to a final volume of 1L and sterilize at 121℃ for 20 minutes. For solid medium, add 1.5% agar powder.

[0059] Test methods

[0060] Construction of CRISPR / Cas9 technology-related vectors

[0061] (1) Construction of pYF2.3G-Ribo-sgRNA::PlIRP1

[0062] Based on the sgRNA design website (http: / / grna.ctegd.uga.edu / ), the target RNA (sgRNA) on the PlIRP1 gene was selected and synthesized by Sangon Biotech. The sgRNA annealing system was prepared according to Table 1, and the annealing was carried out in a metal bath at 37°C for 30 min.

[0063] PlIRP1-sgRNA-F: 5'-CTAGCCAGCGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCGCTGCCAGTTCTTGGTCT-3'

[0064] PlIRP1-sgRNA-R: 5'-AAACAGACCAAGAACTGGCAGCGCGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGGCGCTGG-3'.

[0065] (2) Construction of PYF2.3G-ribo-sgRNA1::NPTII and PYF2.3G-ribo-sgRNA2::NPTII

[0066] Based on the design of the sgRNA website (http: / / grna.ctegd.uga.edu / ), the target RNA (sgRNA, including sgRNA1 and sgRNA2) on the NPTII gene was selected and synthesized by Sangon Biotech. The sgRNA annealing system was prepared according to Table 1, and the annealing was carried out in a metal bath at 37°C for 30 min.

[0067] NPTII-sgRNA1-F: 5'-CTAGCAAGCAGCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCCTGCTTGCCGAATATCATGG-3'

[0068] NPTII-sgRNA1-R:5'-AAACCCATGATATTCGGCAAGCAGGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGCTGCTTG-3'

[0069] NPTII-sgRNA2-F: 5'-CTAGCCAGAGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCTCTGATGCCGCCGTGTTC-3'

[0070] NPTII-sgRNA2-R: 5'-AAACGAACACGGCGGCATCAGAGCGACGAGCTTACTCGTTTCGTCCTCACGGACTCATCAGGCTCTGG-3'.

[0071] Table 1. sgRNA Synthesis System (Takara)

[0072]

[0073] Subsequently, 4 μL of 0.5 mol·L⁻¹ was added to the above system. -1 After mixing with NaCl, boil in a boiling water bath for 2 minutes, then cool at room temperature for 3-4 hours to anneal the DNA fragments and form double strands.

[0074] The target RNA (sgRNA) on the PlIRP1 and NPTII genes was annealed to form a double strand, which was then ligated with the enzyme-digested pYF2.3G-Ribo-sgRNA vector (enzyme digestion system shown in Table 2) using T4-DNA ligase. The ligation system is shown in Table 3. The reaction conditions were 16℃ for 4 h, followed by cooling on ice, and then transformation into E. coli.

[0075] Table 2. Double digestion system of pYF2.3G-Ribo-sgRNA vector (50 μL) (NEB)

[0076]

[0077] Table 3. Ligation reaction system of pYF2.3G-Ribo-sgRNA vector and double-stranded sgRNA (10 μL) (NEB)

[0078]

[0079]

[0080] (3) Construction of pBSSK::PlIRP1 vector

[0081] Based on the approximately 1kb sequences upstream and downstream of the PlIRP1 gene and the NPTII gene, amplification primers were designed (PlIRP1-Left-F, PlIRP1-Left-R, PlIRP1-Right-F, PlIRP1-Right-R, NPTII-F, NPTII-R). PlIRP1-Left-F: 5'-AGAACTAGTGGATCCCCCGGGATCACCGACATGGAGGAGCAG-3'

[0082] PlIRP1-Left-R: 5'-ATCTTGTTCAATCATGTCTGTCGGATGTAGGAGACTGTG-3'

[0083] PlIRP1-Right-F: 5'-GACGAGTTCTTCTGAACGCGCAGAAGGAGAGAAGAG-3'

[0084] PlIRP1-Right-R: 5'-ATCGAATTCCTGCAGCCCGGGCTATCAGGAGCAGGAGACAGCGT-3'

[0085] NPTⅡ-F:5'-ATGATTGAACAAGATGGATTGCAC-3'

[0086] NPTⅡ-R: 5'-TCAGAAGAACTCGTCAAGAAGGC-3'.

[0087] (4) Construction of pBSSK::NPTⅡ vector

[0088] Based on approximately 1 kb sequences upstream and downstream of the PlIRP1 gene, amplification primers (PlIRP1-Left-F, PlIRP1-Right-R) were used to amplify sequences containing the PlIRP1 gene and approximately 1 kb sequences upstream and downstream of it.

[0089] PlIRP1-Left-F: 5'-AGAACTAGTGGATCCCCCGGGATCACCGACATGGAGGAGCAG-3'

[0090] PlIRP1-Right-R: 5'-ATCGAATTCCTGCAGCCCGGGCTATCAGGAGCAGGAGACAGCGT-3'.

[0091] PCR amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozymes). Homologous arm sequences of the left and right arms were amplified using *Phytophthora omycete* genomic DNA as a template. The NPTII gene sequence was amplified from the NPTII resistance vector (reference "Fang Y, Tyler BM. Efficient disruption and replacement of an effector gene in the *Phytophthora omycete* sojae using CRISPR / Cas9[J]. Molecular Plant Pathology, 2016:127-139."). Simultaneously, using *Phytophthora omycete* genomic DNA as a template, sequences containing the PlIRP1 gene and approximately 1 kb upstream and downstream of it were amplified. The specific PCR amplification system is shown in Table 4 below.

[0092] Table 4. Phanta Max high-fidelity enzyme PCR amplification system (50 μL)

[0093]

[0094]

[0095] The amplification program was as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56-72℃ for 15 s, extension at 72℃ for 30 s / kb, for 34 cycles, followed by a final extension of 5 min. The target band was detected by electrophoresis and then purified using an OMEGA agarose gel purification kit; specific steps were followed by referring to the kit's instructions. The obtained product, the left and right homologous arm sequences of the PlIRP1 gene and the NPTII gene concentration, were then ligated using the ClonExpress One Step Cloning Kit (Nanjing Novizan) to a linearized pBSSK vector (circular pBSSK vector digested with SmaI restriction endonuclease NEB at 25℃ for 2 h; the digested product was purified using an OMEGA PCR purification kit). The ligation system is shown in Table 5, yielding pBSSK::PlIRP1. After amplifying the PlIRP1 gene and its upstream and downstream sequences of approximately 1 kb each, this gene sequence can be ligated into a linearized pBSSK vector. The enzyme digestion conditions and ligation system of the pBSSK vector are the same as those of the pBSSK::PlIRP1 vector described above, and finally pBSSK::NPTII is obtained.

[0096] Table 5. Reaction systems for pBSSK::PlIRP1 and pBSSK::NPTII carrier ligation (10 μL)

[0097]

[0098] The constructed pYF2.3G-Ribo-sgRNA::PlIRP1, PYF2.3G-ribo-sgRNA1::NPTII, PYF2.3G-ribo-sgRNA2::NPTII, pBSSK::PlIRP1, pBSSK::NPTII, and pYF2-PsNLS-hSpCas9 were transformed into E. coli.

[0099] (5) E. coli transformation and validation

[0100] 100 μL of E. coli competent cells (DH5α) were freeze-thawed on ice. 10 μL of the ligation product was added, and the mixture was gently tapped to mix. The cells were then incubated on ice for 30 min. A 42°C water shock was performed for 90 s, followed by immediate incubation on ice for 2 min. 650 μL of LB broth was added to the tube, and the cells were incubated at 37°C and 180 rpm for 1 h. During this process, the E. coli recovered its growth state and expressed the plasmid-encoded antibiotic resistance gene. The bacterial suspension was centrifuged at 4000 rpm for 4 min, and the supernatant was collected. The remaining 100 μL of LB broth was used to resuspend the cells, and the suspension was spread onto a plate containing a final concentration of 100 μg / mL. -1 Incubate on Amp's LB solid screening plates at 37°C inverted for 12–16 h.

[0101] Using a single Escherichia coli colony as a template, the validation primers for the pYF2.3G-Ribo-sgRNA vector were M13F and RPL41_Pseq_F; the validation primers for the pBSSK vector were M13F and M13R. Among them, M13F and M13R are universal primers. The sequence of primer RPL41_Pseq_F is as follows: RPL41_Pseq_F: 5'-CAAGCCTCACTTTCTGCTGACTG-3'.

[0102] Colony PCR verification was performed using Green Taq Mix (Nanjing Novozymes), and the system is shown in Table 6 below:

[0103] Table 6. Colony PCR reaction system (20 μL)

[0104]

[0105] The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s / kb, for 30 cycles, followed by a final extension of 7 min. The amplification products were detected by gel electrophoresis. Two colonies with amplified bands matching the size of the target fragment were selected and analyzed using a solution containing 100 μg / mL... -1 Amp was sent for sequencing after being shaken in LB liquid medium.

[0106] (6) Large-scale extraction of plasmid DNA

[0107] Select a single colony of *E. coli* containing the target plasmid for incubation by shaking. Add the single colony to a concentration of 100 μg / mL. -1 Incubate Amp in LB liquid medium at 37°C and 200 rpm for 12 h, then add to 200 mL of LB liquid medium containing Amp and incubate at 37°C and 200 rpm for 14 h.

[0108] The plasmids required for PEG-mediated transformation were extracted using the TIANGEN EndoFree Maxi Plasmid Kit. Three plasmids (pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA::PlIRP1, and pBSSK::PlIRP1) were required for PlIRP1 gene knockout, while four plasmids (pYF2-PsNLS-hSpCas9, pBSSK::NPTII, pYF2.3G-Ribo-sgRNA1::NPTII, and pYF2.3G-Ribo-sgRNA2::NPTII) were required for PlIRP1 gene complementation.

[0109] Example 2: Preparation of Phytophthora litchii protoplasts and PEG-mediated transformation

[0110] Preparation of enzymatic hydrolysate: Weigh 0.15 g of lysing enzyme (SIGMA) and 0.06 g of cellulase (SIGMA) into a sterile beaker, and add 10 mL of 0.8 mol·L⁻¹ hydrolysate into a laminar flow hood. -1 Mannitol, 8 mL sterile ddH2O, 800 μL 0.5 mol·L⁻¹ -1 KCl, 800 μL 0.5 mol·L -1 MES-KOH and 400 μL 0.5 mol·L -1 After fully dissolving CaCl2, transfer it to a 50mL centrifuge tube for later use.

[0111] Preparation of W5 solution: Weigh 7.8g Glucose, 4.6g CaCl2, 2.25g NaCl, and 0.093g KCl, add ddH2O and bring the volume to 250mL. Set aside for later use.

[0112] Preparation of MMg solution: Weigh 18.22g mannitol, 0.76g MgCl2·6H2O, and 2mL MES Buffer, add water to make up to 250mL, and set aside for use;

[0113] Wild-type strain WT of Phytophthora lichei was activated on nutrient pea solid agar (NPB solid medium). Mycelial blocks were placed in Erlenmeyer flasks, and 50 mL of NPB liquid medium was added for cultivation. Three flasks were cultured in the dark at 25°C for 3 days, with shaking every 12 hours. The mycelia were collected by filtration through gauze, gently squeezed with forceps, and added to a 50 mL centrifuge tube containing enzymatic hydrolysate. After gentle mixing, the mycelia were enzymatically hydrolyzed at 25°C and 40 rpm for 40–45 min. After enzymatic hydrolysis, the mycelia were quickly filtered through a 50 mL beaker lined with three layers of Miracloth filter cloth, and the filtrate was transferred to a 50 mL round-bottom centrifuge tube. The tube was centrifuged at 4°C and 1500 rpm for 3 min. The supernatant was discarded, and 10 mL of W5 solution was added to resuspend the protoplasts. Then, 25 mL of W5 solution was added, and the mixture was gently mixed by inverting. The tube was centrifuged at 4°C and 1500 rpm for 4 min. Discard the supernatant, add 7 mL of W5 solution to resuspend the protoplasts, and place on ice for 30 min. Then centrifuge at 1500 rpm for 4 min at 4 °C, discard the supernatant, add 6 mL of MMG solution to resuspend the protoplasts, and place at room temperature for 10 min. Place six sterile 50mL centrifuge tubes on ice. When performing PlIRP1 gene knockout, add 30μg of each of the following plasmids to each centrifuge tube: pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA::PlIRP1, and pBSSK::PlIRP1. When performing PlIRP1 gene complementation, add 30μg of each of the following plasmids to each centrifuge tube: pYF2-PsNLS-hSpCas9, pBSSK::NPTII, pYF2.3G-Ribo-sgRNA1::NPTII, and pYF2.3G-Ribo-sgRNA2::NPTII. This will yield an MMG solution containing protoplasts.

[0114] Add 1 mL of MMG solution containing protoplasts to each 50 mL centrifuge tube, gently swirl to mix, and incubate on ice for 10 min. Add 580 μL of 40% polyethylene glycol (PEG) solution along the wall of each centrifuge tube, repeating three times. During this process, slowly rotate the centrifuge tube to mix the PEG with the protoplasts, and incubate on ice for 20 min. Add 100 mg / mL of pea mannitol culture medium (PM) at a 1000:1 ratio. -1Ampicillin PM was prepared. 2 mL of Amp PM was added to a centrifuge tube, gently inverted, and placed on ice for 2 min. 8 mL of Amp PM was added to the centrifuge tube, gently inverted, and placed on ice for 2 min. Finally, 10 mL of Amp PM was added to each centrifuge tube, gently inverted, and placed at an angle. The tubes were incubated in the dark at 25°C for 14–16 h to allow protoplast regeneration. After overnight incubation, protoplast regeneration was observed under a microscope, followed by centrifugation at 2000 rpm for 5 min. The supernatant was discarded from each centrifuge tube, leaving 5 mL of liquid culture medium. The precipitate was resuspended and 30 mL of medium containing 30 μg / mL of Amp PM was added. -1 Antibiotic-containing pea mannitol solid medium. Genimycin G418 was used for PlIRP1 gene knockout; hygromycin was used for PlIRP1 gene restoration. The corresponding antibiotic was added, mixed thoroughly by inversion, and poured into two 9cm sterile petri dishes. The mixture was incubated in the dark at 25°C for 2–3 days. Single colonies were picked, numbered, and named for identification.

[0115] Example 3: Verification and Measurement

[0116] (1) PCR-agarose gel electrophoresis verification of PlIRP1 gene knockout and complementation transformants

[0117] Genomic DNA was extracted from wild-type *Phytophthora lichee* (WT) and transformants using the CTAB method. Using the genomic DNA as a template, primers PlIRP1-OUT-F and PlIRP1-OUT-R, located outside the left and right homologous arms of PlIRP1 in the genome, were designed for conventional PCR amplification. Band size was detected by gel electrophoresis to verify successful PlIRP1 knockout, and the results were then sent for sequencing.

[0118] PlIRP1-OUT-F:5'-GTCAGAAGGGGTATATGGGGAAAC-3'

[0119] PlIRP1-OUT-R: 5'-GACCTCCTGTATTTGGACACGAAC-3'.

[0120] Sequencing results confirmed that the PEG transformation knockout and complementation were successful, and two PlIRP1 gene knockout transformants and complementation transformants were obtained. They were numbered from T1 according to the verification order. The two successful knockout mutants were named T83 and T123, respectively, and the complementation transformant was named C140.

[0121] (2) Quantitative fluorescence analysis of PlIRP1 gene knockout and complementation transformants to verify

[0122] RNA was extracted from the mycelia of wild-type Phytophthora lichee and transformants using the BBI All-In-One DNA / RNA Mini-Preps Kit. Genomic DNA was removed and cDNA was synthesized by reverse transcription using the Tiangen FastKing cDNA First-Strand Synthesis Kit. Quantitative primers PlActin-F and PlActin-R were designed using the PlActin gene as an internal reference gene, and quantitative primers PlIRP1-qPCR-F and PlIRP1-qPCR-R were designed using the CDS sequence of the PlIRP1 gene as the target gene. Real-time quantitative PCR was performed using the SYBR Premix Ex Taq™ kit (TAKARA). The expression of the PlIRP1 gene was analyzed to verify whether the PlIRP1 gene was successfully knocked out and reintroduced.

[0123] PlActin-F:5'-TCACGCTATTGTTCGTCTGG-3'

[0124] PlActin-R: 5'-TCATCTCCTGGTCGAAGTCC-3'

[0125] PlIRP1-qPCR-F: 5'-AAGTACACGTGGTCCCG-3'

[0126] PlIRP1-qPCR-R: 5'-GCTCGAGTAAAGCGCCAAC-3'.

[0127] (3) Pathogenicity assay of knockout mutants

[0128] Wild-type *Phytophthora lichee* strain WT, the unsuccessfully knocked-out transformant CK grown on G418 antibiotic-containing medium, and the successfully knocked-out mutants T83 and T123, and the complemented transformant C140 were subcultured twice on antibiotic-free CA plates. Mycelial blocks of WT, CK, T83, T123, and C140 with consistent mycelial age were punched and inoculated into the center of 15 mL equal volumes of carrot agar plates (diameter = 9 cm). After incubation at 25℃ in the dark for 5 days, 9 mm diameter mycelial blocks of WT, CK, T83, T123, and C140 with consistent mycelial age were punched again, with 5 blocks from each group placed in 2.5 mL ddH2O and vortexed for 1 min to allow sporangia to fully detach from the sporangiophores, obtaining a sporangium suspension. The sporangium suspension was placed in a 16℃ incubator to allow zoospore release, and the concentration was counted and diluted to 10 zoospores / μL under a microscope. After being washed with ddH2O, litchi fruits (of the Huaizhi variety) were dried on a clean bench. Each litchi fruit was inoculated with 60 μL of zoospore suspension, with 10 fruits inoculated. The fruits were then incubated at 25℃ under humidity. After 4 days, photographs were taken and the diameter of lesions was measured. Differences were analyzed for significance using Duncan's multiple range test in SPSS software.

[0129] Take WT, CK, T83, T123 and C140 mycelial blocks with a diameter of 9 mm obtained from the above subculture and inoculate them on the side of the fruit of Huaizhi litchi variety. Each strain is inoculated on 10 fruits. The fruits are kept moist at 25℃. After 4 days, take pictures and measure the diameter of the lesions.

[0130] (4) Determination of oospore yield in knockout mutants

[0131] After two subcultures, the edges of the colonies of WT, CK, T83, T123 and C140 were removed with a sterile punch to obtain uniformly sized bacterial discs (d=9mm) and transferred to carrot agar plates covered with a Hybond N+ membrane. After 10 days of dark incubation at 25°C, the filter membrane was removed, and four bacterial discs were randomly punched near the inoculation point. The discs were homogenized in 4mL ddH2O, and 1μL was aspirated onto a glass slide to count the number of oospores for each strain.

[0132] Example 4: Results and Analysis

[0133] (1) Construction of the recombinant fragment of the PliIRP1 gene from Phytophthora litchii

[0134] Using PCR technology, we cloned the Left and Right homologous arm sequences of the PlIRP1 gene and the NP TII gene sequence fragment, respectively. These fragments were ligated into a linearized PBSSK vector to successfully obtain the pBSSK::PlIRP1 vector. Double-stranded sgRNA was synthesized and ligated into a linearized pYF2.3G-Ribo-sgRNA vector to successfully obtain the pYF2.3G-Ribo-sgRNA::PlIRP1 vector. A knockout diagram is shown below. Figure 1 .

[0135] (2) Screening of P1IRP1 knockout mutants of Phytophthora litchii

[0136] Genomic DNA was extracted from wild-type *Phytophthora licoricei* WT and transformants using the CTAB method. Using genomic DNA as a template, primers PlIRP1-OUT-F and PlIRP1-OUT-R, located outside the left and right homologous arms of PlIRP1 in the genome, were designed for conventional PCR amplification. Band size was detected by gel electrophoresis. Figure 2 This proves that the PEG conversion knockout was successful.

[0137] Using real-time quantitative PCR primers PlIRP1-qPCR-F and PlIRP1-qPCR-R for the PlIRP1 gene, and PlActin-F and PlActin-R for the PlActin gene as an internal reference gene, the expression of the PlIRP1 gene in knockout transformants was analyzed by real-time quantitative PCR. The results showed that neither transformant T83 nor T123 expressed the PlIRP1 gene, indicating that T83 and T123 were knockout transformants of the PlIRP1 gene. Figure 2 Sequencing results confirmed that it had indeed been knocked out.

[0138] (3) Pathogenicity analysis of PLIRP1 knockout mutants

[0139] Compared with the wild-type WT and CK strains of Phytophthora lichei and the supplementary strain C140, the knockout mutants T83 and T123, after inoculation with the same concentration of zoospore suspension and mycelial cakes with the same colony diameter, showed a significant reduction in lesion diameter, demonstrating that the deletion of the PlIRP1 gene significantly reduces the pathogenicity of Phytophthora lichei. Figure 3 , Figure 4 ).

[0140] (4) Effect of gene PlIRP1 knockout mutant on oospore yield

[0141] Oospore suspensions of wild-type WT, CK, and complement strain C140 and knockout mutants T83 and T123 were prepared under the same treatment conditions. Compared with WT, CK, and complement strain C140, the ospore yield of knockout mutants T83 and T123 was decreased, indicating that the knockout of the gene PlIRP1 affects the production of Phytophthora downy mildew oospores. Figure 5 ).

[0142] Therefore, the gene provided by this invention can be used for the prevention and control of plant diseases, particularly litchi downy mildew caused by Phytophthora licheniformis. Furthermore, the gene provided by this invention can serve as a drug target for the prevention and control of plant diseases. Those skilled in the art can develop drugs for the prevention and control of plant diseases, especially litchi downy mildew, based on the guidance and inspiration provided in this specification.

[0143] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent alternatives and are included within the protection scope of the present invention.

Claims

1. A licorice downy mildew-associated protein PlIRP1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

2. The biomaterial related to the pathogenic protein P1IRP1 of Phytophthora licheeis as described in claim 1, characterized in that: It can be any one of the following biological materials: 1) The nucleic acid molecule encoding the pathogenic protein PlIRP1 of Phytophthora downy mildew; 2) An expression cassette containing the nucleic acid molecules described in 1); 3) A recombinant vector containing the expression cassette described in 2); 4) Recombinant microorganisms containing the recombinant vector described in 3).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule mentioned in 1) is the gene sequence of the litchi downy mildew pathogenicity-associated protein PlIRP1, as shown in SEQ ID NO:

1.

4. The biomaterial related to the pathogenic protein P1IRP1 of Phytophthora licheeis as described in claim 1, characterized in that: It can be any one of the following biological materials: 5) Knock out the sgRNA of the gene of the downy mildew-associated protein PlIRP1; the sgRNA is as follows: 5'-CTAGCCAGCGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCGCTGCCAGTTCTTGGTCT-3'; 6) A gene knockout vector prepared using the sgRNA described in 5) to inhibit or block the gene expression of the litchi downy mildew-associated protein PlIRP1; 7) The gene-deficient Phytophthora litchii, which is a pathogenic protein related to Phytophthora litchii, prepared using the gene knockout vector described in 6).

5. The application of the litchi downy mildew-associated protein P1IRP1 as described in claim 1, characterized in that: For any of the following applications: i) The application of knocking out the pathogenicity-associated protein PlIRP1 of Phytophthora lichee in reducing the pathogenicity of Phytophthora lichee; ii) The application of knocking out the pathogenic protein PlIRP1 of Phytophthora lichee in reducing the infectivity of Phytophthora lichee; iii) The application of knocking out the pathogenic protein PlIRP1 of Phytophthora litica in reducing the formation of Phytophthora litica oospores; iv) The application of knocking out the litchi downy mildew-associated protein PlIRP1 in the prevention and control of litchi downy mildew.

6. A method for preventing and controlling litchi downy mildew caused by Phytophthora licheniformis, characterized in that: This is achieved by knocking out the gene of P1IRP1, the pathogenic protein associated with Phytophthora litica as described in claim 1.

7. A method for constructing a *Phytophthora litica* mutant with PlIRP1 gene knockout, characterized in that: Includes the following steps: (1) Using the gene sequence of the litchi downy mildew-associated protein PlIRP1 as described in claim 1, sgRNA was designed using the sgRNA website, and the sgRNA was ligated to the pYF2.3G-Ribo-sgRNA vector to obtain... PlIRP1 Gene knockout plasmid pYF2.3G-Ribo-sgRNA:: PlIRP1 ; Alternatively, primers for amplifying the left and right homologous arms can be designed based on the gene sequence of the pathogenic protein PlIRP1 of Phytophthora licoriceis as described in claim 1. The left and right homologous arms can be amplified using Phytophthora licoriceis genomic DNA as a template. PlIRP1 Left and right homologous arms of the gene and NPTⅡ The gene fragment was ligated into the pBSSK vector to obtain... PlIRP1 Gene knockout plasmid pBSSK:: PlIRP1 ; (2) PlIRP1 Gene knockout plasmid pYF2.3G-Ribo-sgRNA:: PlIRP1 or pBSSK:: PlIRP1 After introducing the strain into the protoplast of wild-type Phytophthora licoriceis, and through screening and verification, the following results were obtained. PlIRP1 The gene knockout mutant is the litchi downy mildew mutant with the PLIRP1 gene knocked out. Left homologous arm amplification primers: PlIRP1 -Left-F:5'-AGAACTAGTGGATCCCCCGGGATCACCGACATGGAGGAGCAG-3'; PlIRP1 -Left-R:5'-ATCTTGTTCAATCATGTCTGTCGGATGTAGGAGACTGTG-3'; Right homologous arm amplification primers: PlIRP1 -Right-F:5'-GACGAGTTCTTCTGAACGCGCAGAAGGAGAGAAGAG-3'; PlIRP1 -Right-R:5'-ATCGAATTCCTGCAGCCCGGGCTATCAGGAGCAGGAGACAGCGT-3'。 8. The method for constructing the *Phytophthora litchi* mutant with PlIRP1 gene knockout according to claim 7, characterized in that: The sgRNA mentioned in step (1) is shown below: sgRNA: 5'-CTAGCCAGCGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCGCTGCCAGTTCTTGGTCT-3'.

Images