Gene plswl1 and application thereof in preventing and treating li-chi downy mildew

Abstract

This invention discloses genes PlSWL1 And its application in the prevention and control of downy mildew in litchi. This invention constructs... PlSWL1 The gene was knocked out via vector and transformed into a protoplast using PEG-mediated homologous recombination. PlSWL1 Knockout mutants and replacement transformants were obtained from *Phytophthora licoricei* through knockout and replacement. Compared with the wild type, the mutants showed a significantly reduced germination rate of dormant spores and a markedly weakened pathogenicity of zoospores and hyphae. This invention confirms... PlSWL1 It is essential for the growth, development, infection, and pathogenicity of Phytophthora lichei, and helps to elucidate the pathogenic molecular mechanism of Phytophthora lichei, providing a target gene for the development of effective fungicides.

Description

Technical Field

[0001] This invention belongs to the field of green prevention and control technology for crop diseases, specifically involving the gene PlSWL1 and its application in the prevention and control of downy mildew in litchi. Background Technology

[0002] The disease caused by *Peronophythora litchii* Chen ex Ko et al. is litchi downy mildew, and its host plant is litchi. This pathogen belongs to the subphylum Oomycetes, class Oomycetes, order Peronosporales, and family Peronophthoraceae. Litchi downy mildew caused by *Peronophythora litchii* severely restricts the development of the litchi industry, and no disease-resistant litchi varieties have yet been developed. Currently, the control of litchi downy mildew still relies mainly on pesticides, but the potential for pesticide resistance and food safety issues cannot be ignored. Therefore, studying the pathogenic mechanism of *Peronophythora litchii* is of great significance.

[0003] Studies have shown that downy mildew damages litchi mainly relies on a series of pathogenic factors. Analyzing the functions of pathogenic genes related to litchi is of practical significance for controlling downy mildew and breeding disease-resistant varieties.

[0004] The sugar transporter encoded by PlSWL1 belongs to the SWEET protein family (Sugars Will Eventually Be Exported Transporters, or SWEET for short). SWEET is a class of sugar transporters widely distributed in the biological world. It can efficiently mediate the transmembrane transport of sugars (such as glucose, fructose, and sucrose) and participate in the distribution of sugars between cells. It can affect the growth and development of organisms. Investigating its role in the growth and development of Phytophthora litchii is expected to provide a theoretical basis and molecular foundation for screening novel targets for the control of Phytophthora litchiii and developing green control strategies that intervene in sugar metabolism / transportation-related processes. Summary of the Invention

[0005] In view of the defects or deficiencies in the existing technology, the primary objective of this invention is to provide a litchi downy mildew-related protein, PlSWL1.

[0006] Another objective of this invention is to provide biomaterials related to the above-mentioned lychee downy mildew pathogenicity-associated protein PlSWL1.

[0007] Another object of the present invention is to provide the application of the above-mentioned litchi downy mildew pathogenicity-related protein PlSWL1 or biomaterials.

[0008] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0009] This invention discloses a previously unknown protein, PlSWL1, and its encoding gene, PlSWL1, from *Phytophthora licoricei*. The full-length and CDS sequence of the PlSWL1 gene are shown in SEQ ID NO.1, encoding 265 amino acids, as shown in SEQ ID NO.2. This invention utilizes CRISPR / Cas9 gene editing technology to construct pBSSK::NPTⅡ, PYF2.3G-ribo-sgRNA1::PlSWL1, and PYF2.3G-ribo-sgRNA2::PlSWL1 knockout vectors, and knocks out the PlSWL1 gene using PEG-mediated protoplast transformation. Knockout mutants T4, T5, and T40 (named according to the transformant verification sequence) and one complement transformant C were obtained. All three mutants exhibited significant defects in the pathogenicity of *Phytophthora licoricei*. Pathogenicity assays showed that the knockout mutants T4, T5, and T40 of the PlSWL1 gene significantly reduced pathogenicity in young litchi leaves (variety: Guiwei). The above experiments demonstrate that the PlSWL1 gene of Phytophthora litchii is a pathogenic gene associated with Phytophthora litchiii.

[0010] A litchi downy mildew-associated protein PlSWL1, the amino acid sequence of which is 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.

[0011] The aforementioned biological materials related to the PlSWL1 protein, a pathogenic protein associated with Phytophthora downy mildew of litchi, are any one or more combinations of the following biological materials:

[0012] 1) The nucleic acid molecule encoding the plSWL1 protein, which is associated with the pathogenicity of Phytophthora licheeis;

[0013] 2) An expression cassette containing the nucleic acid molecules described in 1);

[0014] 3) A recombinant vector containing the expression cassette described in 2);

[0015] 4) Recombinant microorganisms containing the recombinant vector described in 3);

[0016] 5) Nucleic acid molecules that inhibit or block the gene expression of the litchi downy mildew-associated protein PlSWL1;

[0017] 6) A 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 PlSWL1;

[0018] 7) The gene-deficient Phytophthora litchii pathogenicity-related protein PlSWL1 was prepared using the gene knockout vector described in 6).

[0019] Further, the nucleic acid molecule mentioned in 1) is the gene sequence or CDS sequence of the litchi downy mildew pathogenicity-associated protein PlSWL1, 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.

[0020] Furthermore, the nucleic acid molecule mentioned in 5) is the antisense RNA, siRNA, shRNA, or sgRNA of the PlSWL1 gene.

[0021] Furthermore, the sgRNA may be any of the following sequences:

[0022] PlSWL1-sgRNA1: 5'-CGCTGTCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCACAGCGATTGAGCCCAGTAC-3';

[0023] PlSWL1-sgRNA2: 5'-CACAATCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCATTGTGGTGCCGAATATTGT-3'.

[0024] The application of the aforementioned litchi downy mildew-associated protein PlSWL1 or biological materials is any one or more combinations of the following applications:

[0025] i) Application in regulating the pathogenicity of Phytophthora downy mildew on litchi;

[0026] ii) Application in regulating the expression levels of genes related to downy mildew in litchi;

[0027] iii) Application in regulating the formation of oospores of Phytophthora downyensis on litchi;

[0028] iv) Application in the prevention and control of lychee downy mildew;

[0029] v) Application as a target in the design and screening of drugs against downy mildew of litchi.

[0030] 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 PlSWL1 of Phytophthora licheeis.

[0031] 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 PlSWL1.

[0032] The method for constructing the above-mentioned drug screening model against Phytophthora licheniformis includes the following steps:

[0033] (1) Design sgRNA based on the PlSWL1 gene sequence, and ligate the sgRNA with the pYF2.3G-Ribo-sgRNA vector to obtain the PlSWL1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlSWL1;

[0034] And / or, based on the 1kb upstream and downstream sequences of the PlSWL1 gene sequence, primers for amplifying the left and right homologous arms were designed. Using Phytophthora litchii genomic DNA and NPTII synthetic fragments as templates, the left and right homologous arms and NPTII were amplified and ligated with the pBSSK vector to obtain the PlSWL1 gene knockout plasmid pBSSK::NPTII.

[0035] (2) The PlSWL1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlSWL1 and / or pBSSK::NPTⅡ were introduced into the protoplasts of wild-type strain of Phytophthora litchii. After screening and verification, the PlSWL1 gene knockout mutant was obtained, which is the drug screening model of Phytophthora litchii.

[0036] Furthermore, the sgRNA described in step (1) can be any of the following sequences:

[0037] PlSWL1-sgRNA1: 5'-CGCTGTCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCACAGCGATTGAGCCCAGTAC-3';

[0038] PlSWL1-sgRNA2: 5'-CACAATCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCATTGTGGTGCCGAATATTGT-3'.

[0039] Furthermore, the amplification primers for the left and right homologous arms mentioned in step (1) are as follows:

[0040] Left homologous arm amplification primers:

[0041] PlSWL1-Left-F: 5'-AATTCCTGCAGCCCACTCCATGACATCGCAGTACTTGTGAG-3';

[0042] PlSWL1-Left-R: 5'-TCCATCTTGTTCAATCATTTTGAAATAGCTGTGGACCGGGTAC-3';

[0043] Right homologous arm amplification primers:

[0044] PlSWL1-Right-F: 5'-TAGAACTAGTGGATCCCCCTCGAGCGTTTTGTGGTGGGTC-3';

[0045] PlSWL1-Right-R: 5'-ACGAGTTCTTCTGACGAGAACTACAGCCATCTAACTAAGAT-3'.

[0046] The present invention has the following advantages and effects compared with the prior art:

[0047] This invention demonstrates through experiments that amplifying the upstream and downstream sequences of the gene PlSWL1, linking them to the vector pBSSK (a generous donation from the Laboratory of Oomycetes and Fungi Molecular Biology, College of Plant Protection, Nanjing Agricultural University), and then performing homologous recombination followed by polyethylene glycol (PEG)-mediated protoplast transformation and CRISPR / Cas9 knockout, yielded mutants. Compared to the wild type, PlSWL1 was found to affect the mycelial growth rate, resting spore germination rate, and pathogenicity of *Phytophthora litchifolia*, indicating that this gene is related to the growth, development, and maintenance of pathogenicity of the pathogen. This invention confirms that the gene PlSWL1 is essential for the pathogenicity 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

[0048] Figure 1 This is a schematic diagram of the construction of homologous recombination of the PlSWL1 gene from Phytophthora litchii.

[0049] Figure 2 This is a PCR amplification result of Phytophthora licoricei PlSWL1 gene knockout transformants; where lane WT: wild type Phytophthora licoricei; lane CK: transformants whose knockout was unsuccessful; lane C: PlSWL1 gene complementation transformants; lanes T4, T5, and T40 represent three knockout transformants of the PlSWL1 gene, respectively.

[0050] Figure 3 These are the mycelial growth rate results for wild-type WT, CK, C and knockout mutants T4, T5, T40. a is a mycelial morphology diagram; b is a statistical result diagram of growth rate.

[0051] Figure 4 The graph shows the statistical results of the germination rate of dormant spores for wild-type WT, CK, C and knockout mutants T4, T5, T40; where a is the morphological diagram of the dormant spores and b is the statistical result of the germination rate of the dormant spores.

[0052] Figure 5The images show disease lesions on litchi leaves after inoculation with mycelial block suspensions of wild-type WT, CK, C and knockout mutants T4, T5, T40; where b is the statistical area of ​​lesion a, c is the disease lesion on litchi leaves after inoculation with zoospores of WT, CK, C and PlSWL1 mutants, and d is a bar chart of the statistical area of ​​lesion c. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0054] 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.

[0055] Nucleotide sequence of the Phytophthora licoricei PlSWL1 gene (SEQ ID NO.1):

[0056] ATGGTGTCTGCGTTCGTCTTAACGATCAAAATATTGACAACTATTGCTCAAATTGCACAGCGATTGAGCCCAGTACCGGATCTGTATCGCGTTCACAAACAACGGGACACCGGAGTTATGGCCTTCATACCATTAGTCGCCTTGCTGTTGGGCAACCATATCTGGTAAGATAAATTCTCTCTTGGGGCAAACCCTCAGTATTGGTTTCACTAATTTCTAATTTTTTTGGTACCAGGTTACTTTACGCCTACGTCGTTGAGAATATATTCCCACTATTCGCGGTGTGTGCCTTTGGTGACATCATCTTAGCCATCTACATTGTGATTTACGCAAAATATTGCCCAAACCGAGCGTATGTGGTGAAGACCATTGTCATGGGGGCAATTCCGTTCGTGTTGGTTTCTGCGTACGCCATTTTGGTGGCTGTGAAGGCGATCGACCAGTCAAGAAGTCAATTTGGCGACATACTCGGGTACTTGGGAAACGCGGCGATGTTCGCCTTGTACTTTTCGCCGTTTGAGAAGATCAAGCTCGTGATCGAGACCAAATCATCAGCTGCAATCCCCGTACTTCTATGTGGAATTATCTTCGTCAACAGTAGCTTGTGGCTCATCAACGGAATCATCGACAACGACTTGTTTATTGTGGTGCCGAATATTGTAGGGGTTACTCTGACTGCAGTCCAGCTTATGTTGTATTACATTTATCGCCCAACTAGGCACACTTCATCGGTGGAAACAGGCGGCAGTGAACTTGATCTTGTGGATCTGGAAATGAGCACAACCGCAAAAGTTTGGTCAGCAAATAATTCTGCGTTTGCTGCTCTGGTATCACCAAAAACTCCAACGAAGCAAGAAGTATTTTAA。

[0057] Amino acid sequence of the pathogenicity-related protein PlSWL1 of Peronophythora litchii (SEQ ID NO.2):

[0058] MVSAFVLTIKILTTIAQIAQRLSPVPDLYRVHKQRDTGVMAFIPLVALLLGNHIWLLYAYVVENIFPLFAVCAFGDIILAIYIVIYAKYCPNRAYVVKTIVMGAIPFVLVSAYAILVAVKAIDQSRSQFGDI LGYLGNAAMFALYFSPFEKIKLVIETKSSAAIPVLLCGIIFVNSSLWLINGIIDNDLFIVVPNIVGVTLTAVQLMLYYIYRPTRHTSSVETGGSELDLVDLEMSTTAKVWSANNSAFAALVSPKTPTKQEV.

[0059] Example 1: Construction of the PlSWL1 gene knockout vector

[0060] Test strains, plants, and vectors:

[0061] The tested strains were *Peronophythora litchii* (Wild Type, WT), a common *Phytophthora* species, which can be obtained commercially or naturally; and *Escherichia coli* strain JM109, which can be obtained commercially. The inoculated plant material was young leaves of *Litchi chinensis* (Guiwei variety) (collected from the horticultural practice orchard of South China Agricultural University). 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 "A CRISPR / Cas9‐mediated in situcomplementation method for *Phytophthora sojae* mutants[J]. Molecular Plant Pathology, The above vectors were kindly provided by the Laboratory of Oomycetes and Fungi Molecular Biology, College of Plant Protection, Nanjing Agricultural University. (Published in 2021, 22(3)).

[0062] Main test culture media:

[0063] Carrot agar (CA) (1 L): Juice 300 g of carrots, filter through gauze, and sterilize at 121°C for 20 min. Add 1.5% (w / v) agar powder to the solid medium.

[0064] LB medium (1 L): 5 g yeast extract, 10 g tryptone, 10 g sodium chloride (NaCl), sterilized at 121℃ for 20 min. For solid medium, add 1.5% (w / v) agar powder.

[0065] Nutrition Pea Broth (NPB) (1 L): Boil 120 g of fresh peas in water for 20 min, then filter. Add 5 g of D-Sorbitol, 5 g of D-Mannitol, 5 g of glucose, 3 g of potassium nitrate (KNO3), 2 g of calcium carbonate (CaCO3), 2 g of yeast extract, and 1 g of dipotassium hydrogen phosphate (K2HPO4) to the filtrate. 4） 1 g potassium dihydrogen phosphate (KH2PO4), 0.5 g magnesium sulfate (MgSO4), 0.1 g calcium chloride (CaCl2), 2 mL vitamin stock solution and 2 mL trace elements were added to a final volume of 1 L with ddH2O and sterilized at 121°C for 20 min. For solid culture media, 1.5% (w / v) Difco Bacto Agar was added.

[0066] Pea mannitol medium (Pea / 0.5 mol∙L) -1 Manitol (PM) (1 L): Boil 120 g of fresh peas in water for 20 min, then filter. Add 91 g D-Mannitol, 2 g CaCO3 and 1.32 g CaCl2 to the filtrate, and bring the volume to 1 L with ddH2O. Sterilize at 121°C for 20 min. For solid culture medium, add 1.5% (w / v) Difco Bacto Agar.

[0067] Piper's medium (1 L): 0.5 g potassium dihydrogen phosphate, 0.25 g magnesium sulfate heptahydrate (MgSO4•7H2O), 1 g L-asparagine, 1 mg vitamin B1, 0.5 g yeast extract, 10 mg β-sitosterol, and 5 g glucose. Add ddH2O to a final volume of 1 L and sterilize at 121°C for 20 min. For solid medium, add 1.5% agar powder.

[0068] Lima bean culture medium: Soak 120 g of lima beans in water for 1 day, add 2 L of deionized water, boil at 121°C, filter through gauze to remove residue, and add deionized water to bring the volume to 2 L. When preparing solid culture medium, in addition to the above reagents, add 1.5% agar powder. Sterilize at 121°C.

[0069] Construction of CRISPR / Cas9 technology-related vectors:

[0070] (1) Construction of pYF2.3G-Ribo-sgRNA::PlSWL1

[0071] Based on the sgRNA design website (http: / / grna.ctegd.uga.edu / ), the target RNA (sgRNA) on the PlSWL1 gene was selected, and after being synthesized by Sangon Biotech, the sgRNA amplification system was prepared according to Table 1, and the amplification procedure is shown in Table 2.

[0072] The sgRNA sequence is as follows:

[0073] PlSWL1-sgRNA1: 5'-CGCTGTCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCACAGCGATTGAGCCCAGTAC-3';

[0074] PlSWL1-sgRNA1-F: 5'-ATTCGTTCGGTGACGCTAGCCGCTGTCTGATGAGTCCGTG-3';

[0075] PlSWL1-sgRNA1-R: 5'-GCTATTTCTAGCTCTAAAACGTACTGGGCTCAATCGCTGT-3';

[0076] PlSWL1-sgRNA2: 5'-CACAATCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCATTGTGGTGCCGAATATTGT-3';

[0077] PlSWL1-sgRNA2-F: 5'-ATTCGTTCGGTGACGCTAGCCACAATCTGATGAGTCCGTG-3';

[0078] PlSWL1-sgRNA2-R: 5'-GCTATTTCTAGCTCTAAAACACAATATTCGGCACCACAAT-3';

[0079] NPTⅡ-sgRNA1: 5'-CAGAGCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGCTCTGATGCCGCCGTGTTC-3';

[0080] NPTⅡ-sgRNA1-F: 5'-ATTCGTTCGGTGACGCTAGCAGAGCCTGATGAGTCCGTG-3';

[0081] NPTⅡ-sgRNA1-R: 5'-GCTATTTCTAGCTCTAAAACGAACACGGCGGCATCAGAGC-3';

[0082] NPTⅡ-sgRNA2: 5'-AATATCCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCGATATTGCTGAAGAGCTTGG-3';

[0083] NPTⅡ-sgRNA2-F: 5'-ATTCGTTCGGTGACGCTAGCAATATCCTGATGAGTCCGTG-3';

[0084] NPTⅡ-sgRNA2-R: 5'-GCTATTTCTAGCTCTAAAACCCAAGCTCTTCAGCAATATC-3'.

[0085] Table 1. sgRNA amplification system (50 µL) (Takara)

[0086]

[0087] Table 2 sgRNA amplification program

[0088]

[0089] The target RNA (sgRNA) on the PlSWL1 gene was amplified to form a double strand. After detection by electrophoresis, the target band was recovered using an OMEGA agarose gel purification and recovery kit. The specific steps were as per the kit's instructions. The target RNA 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 12 h, followed by cooling on ice, and then transformation into E. coli.

[0090] Table 3. Double digestion system of pYF2.3G-Ribo-sgRNA vector (50 μL) (NEB)

[0091]

[0092] Table 4. Ligation reaction system of pYF2.3G-Ribo-sgRNA vector and double-stranded sgRNA (10 μL) (NEB)

[0093]

[0094] (2) Construction of pBSSK::NPTⅡ and pBSSK::PlSWL1 and vectors

[0095] Based on the approximately 1kb sequence upstream and downstream of the PlSWL1 gene, left and right homologous arms and amplification primers (PlSWL1-Left-F, PlSWL1-Left-R, PlSWL1-Right-F, PlSWL1-Right-R) were designed.

[0096] PlSWL1-Left-F: 5'-AATTCCTGCAGCCCACTCCATGACATCGCAGTACTTGTGAG-3';

[0097] PlSWL1-Left-R: 5'-TCCATCTTGTTCAATCATTTTGAAATAGCTGTGGACCGGGTAC-3';

[0098] PlSWL1-Right-F: 5'-TAGAACTAGTGGATCCCCCTCGAGCGTTTTGTGGTGGGTC-3';

[0099] PlSWL1-Right-R: 5'-ACGAGTTCTTCTGACGAGAACTACAGCCATCTAACTAAGAT-3'.

[0100] Amplification primers (NPTII-F, NPTII-R) were designed based on the NPTII gene sequence:

[0101] NPTⅡ-F: 5'-ATGATTGAACAAGATGGATT-3';

[0102] NPTⅡ-R: 5'-TCAGAAGAACTCGTCAAGAA-3';

[0103] Amplification primers (PlSWL1-F, PlSWL1-R) were designed based on the PlSWL1 gene sequence:

[0104] PlSWL1-F: 5'-ATGGTGTCTGCGTTCGTCTTAACGATCAAAATATTG-3';

[0105] PlSWL1-R: 5'-ACCAAAAACTCCAACGAAGCAAGAAGTATTTTAA-3'.

[0106] PCR amplification was performed using the high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novozymes). The left and right homologous arms and PlSWL1 were amplified using Phytophthora litchii genomic DNA as a template. The specific PCR amplification system is shown in Table 4 below.

[0107] Table 5 Phanta Max high-fidelity enzyme PCR amplification system (50 μL)

[0108]

[0109] 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 recovered using an agarose gel purification kit from OMEGA after electrophoresis; specific steps were described in the kit's instructions. After determining the product concentration, it was ligated to a linearized pBSSK vector (enzyme digestion system shown in Table 5) using the ClonExpress One Step Cloning Kit (Nanjing Novizan). The ligation system is shown in Table 6.

[0110] Table 6 pBSSK enzyme digestion reaction system (10 µL)

[0111]

[0112] Table 7. Reaction system for pBSSK::NPTⅡ or pBSSK::PlSWL1 carrier linkage (10 μL)

[0113]

[0114] (3) Transformation and validation of Escherichia coli

[0115] 100 μL of E. coli competent cells JM109 were freeze-thawed on ice. 10 μL of 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 applied 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. 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. -1Incubate on Amp's LB solid screening plates at 37°C for 12–16 h.

[0116] 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'.

[0117] Colony PCR verification was performed using Green Taq Mix (Nanjing Novozymes), and the system is shown in Table 8 below:

[0118] Table 8. Colony PCR reaction system (20 μL)

[0119]

[0120] 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 34 cycles, followed by a final extension of 7 min. The amplified 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. -1 Amp was sent for sequencing after being shaken in LB liquid medium.

[0121] (4) Large-scale extraction of plasmid DNA

[0122] Select a single E. coli colony containing the target plasmid for incubation by shaking. Add the single colony to a concentration of 100 μg·mL⁻¹. -1 Incubate Amp in LB liquid medium at 37°C and 180 rpm for 12 h, then add to 200 mL of LB liquid medium containing Amp and incubate at 37°C and 180 rpm for 14 h.

[0123] The following plasmids were extracted for PEG-mediated transformation using the TIANGEN EndoFree Maxi Plasmid Kit: pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA1::PlSWL1, pYF2.3G-Ribo-sgRNA2::PlSWL1, pYF2.3G-Ribo-sgRNA1::NPTⅡ, pYF2.3G-Ribo-sgRNA2::NPTⅡ, pBSSK::PlSWL1, and pBSSK::NPTⅡ.

[0124] Example 2: Preparation of Phytophthora litchii protoplasts and PEG-mediated transformation

[0125] 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 solution into a laminar flow hood. -1 Mannitol, 8 mL sterile ddH2O, 800 μL 0.5 mol∙L -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 50 mL centrifuge tube for later use.

[0126] Preparation of W5 solution: Weigh 7.8 g Glucose, 4.6 g CaCl2, 2.25 g NaCl, and 0.093 g KCl, add ddH2O and bring the volume to 250 mL, then set aside for use;

[0127] 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;

[0128] 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 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 the protoplasts were resuspended in 10 mL of W5 solution. 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 50 mL centrifuge tubes on ice. When performing PlSWL1 gene knockout, add 30 μg each of the following plasmids to each centrifuge tube: pYF2-PsNLS-hSpCas9, pYF2.3G-Ribo-sgRNA1::PlSWL1, pYF2.3G-Ribo-sgRNA2::PlSWL1, and pBSSK::NPTⅡ. When performing PlSWL1 gene complementation, add 30 μg each of the following plasmids to each centrifuge tube: pYF2.3G-Ribo-sgRNA1::NPTⅡ, pYF2.3G-Ribo-sgRNA2::NPTⅡ, and pBSSK::PlSWL1. This will yield an MMG solution containing protoplasts.

[0129] 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⁻¹ to pea mannitol culture medium (PM) at a ratio of 1000:1. -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 to the remaining 5 mL of liquid culture medium. After resuspending the precipitate, 30 mL of medium containing 30 μg·mL⁻¹ was added. -1 Genimycin G418 was mixed thoroughly with pea mannitol on solid medium by inverting and then poured into two 9 mm 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.

[0130] Example 3: Verification and Measurement

[0131] (1) Validation analysis of PlSWL1 gene knockout transformants

[0132] Genomic DNA was extracted from wild-type Phytophthora licoricei (WT) and transformants using the CTAB method. Using the genomic DNA as a template, primers PlSWL1BW-F and PlSWL1BW-R, located outside the left and right homologous arms of PlSWL1 in the genome, were designed for conventional PCR amplification. Band size was detected by gel electrophoresis to verify successful PlSWL1 knockout, and the results were then sent for sequencing.

[0133] PlSWL1-BW-F: 5'-TCCATGATAGTACATGAAACCTAGATATGCA-3';

[0134] PlSWL1-BW-R: 5'-TCCATGAGTTTGAATAACGCACTGACAG-3'.

[0135] Sequencing results confirmed that the PEG transformation knockout and complementation were successful, and three PlSWL1 gene knockout transformants and one complementation transformant were obtained. The knockout transformants were numbered sequentially starting from T1. The three successfully knocked-out mutants were named T4, T5, and T40, respectively; the complementation transformant was named C.

[0136] (2) Sporangium suspensions of each strain were obtained according to the above method, and zoospores were released after standing at 16℃ for 1 h. The sporangium suspensions were filtered through a nylon membrane with a pore size of 20 μm to obtain zoospore suspensions. The zoospore flagella were removed by vortexing for 1 min. The resting spore suspensions were placed in an incubator at 26℃ and incubated at 60 rpm for 2 h. 1 μL was taken onto a glass slide and observed under a microscope to count the number of resting spores germinating per 100 resting spores. Three independent replicate experiments were set up and the data were analyzed for significance. Pathogenicity determination of knockout mutants

[0137] (3) Pathogenicity determination of mycelial blocks and zoospores

[0138] Lychee (variety: Guiwei) tender leaves were soaked in ddH2O and then placed on moist filter paper. Mycelial blocks and zoospores of 10 mm diameter (WT, C, T4, T5, T40) of uniform age, obtained from subculture on antibiotic-free medium, were inoculated, with the mycelial growth surface covering the underside of the leaf. A zoospore suspension of 10 spores / μL was then inoculated onto the underside of the leaf. Six tender leaves of similar age were repeatedly inoculated for each strain. The leaves were kept at 25℃ and kept moist. After 48 h, photographs were taken and the lesion area was calculated. Difference significance analysis was performed using Duncan's multiple range test in SPSS software.

[0139] Example 4: Results and Analysis

[0140] (1) Construction of the recombinant fragment of Phytophthora licoricei PlSWL1 gene

[0141] The Left and Right homologous arms of the PlSWL1 gene were cloned using PCR technology. Multiple fragments were ligated into a linearized pBSSK vector to successfully obtain the pBSSK::NPTⅡ vector. Double-stranded sgRNA was synthesized and ligated into a linearized pYF2.3G-Ribo-sgRNA vector to successfully obtain the pYF2.3G-Ribo-sgRNA1::PlSWL1 and pYF2.3G-Ribo-sgRNA2::PlSWL1 vectors. A knockout diagram is shown below. Figure 1 The same applies to replenishing the carrier.

[0142] (2) Screening of Phytophthora licoricei PlSWL1 knockout mutant

[0143] The left and right outer primers for the PlSWL1 gene were designed as PlSWL1-BW-F / R. DNA was extracted from wild-type (WT), complemented transformant (C), and PlSWL1 gene knockout mutants of Phytophthora licoriceis. PCR amplification was performed using the wild-type as a control. The amplification method and system were as described in Example 1. The results showed that the fragment amplified using the outer primers was approximately 3000 bp, and the successfully knocked-out fragment was approximately 2900 bp. This further confirms that T4, T5, and T40 are knockout mutants of the PlSWL1 gene. Figure 2 Sequencing results confirmed that the knockout had indeed occurred. The sequencing results for T4, T5, and T40 are as follows:

[0144]

[0145] (3) Analysis of mycelial growth rate of Phytophthora licoricei PlSWL1 knockout mutant

[0146] Using a sterile punch (d = 7 mm), fresh mycelial cakes of the test strains of the same age (PlSWL1 knockout mutant, WT, CK, and C) were collected and inoculated into the center of CA agar plates. The plates were incubated in the dark at 26°C for 7 days, photographed, and their colony diameters were recorded. The growth rate was calculated. Three independent replicate experiments were conducted, and the data were analyzed for significance. The growth rate was calculated as growth rate = colony diameter / number of days. The results showed (…). Figure 3 ): WT: 12.89 mm / d, CK: 12.36 mm / d, T4: 9.78 mm / d, T5: 9.75 mm / d, T40: 9.82 mm / d, C: 12.56 mm / d. Statistical results show that knockout of PlSWL1 significantly affects the growth of Phytophthora litchii.

[0147] (4) Germination analysis of dormant spores of Phytophthora licoricei PlSWL1 knockout mutant

[0148] Sporangium suspensions of wild-type WT, CK, C, and PlSWL1 knockout mutants after two subcultures were obtained. These suspensions were then shaken to prepare resting spore suspensions. The resting spore suspensions were treated at 26℃, 60 rpm for 2 h, followed by the addition of 2 μL of 4% CuSO4. 2 μL of each suspension was observed and the resting spore germination rate was calculated under a microscope. The experiment was repeated three times. The statistical results were: WT: 80.56%, CK: 81.33%, T4: 20.69%, T5: 17.23%, T40: 16.76%, C: 82.76% (…). Figure 4 According to the statistical results, the germination rate of plSWL1 knockout mutants was significantly different from that of WT and CK, and the phenotype of C was restored. Therefore, plSWL1 knockout affects the germination of lychee downy mildew spores.

[0149] (5) Pathogenicity analysis of Phytophthora licoricei PlSWL1 knockout mutant

[0150] Compared with wild-type WT and complemented transformant C, the knockout mutants T4, T5, and T40 showed significantly reduced pathogenicity to litchi leaves, regardless of whether zoospores or mycelial blocks were inoculated. Figure 5 ).

[0151] Experimental results demonstrate that 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.

[0152] 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 pathogenic protein PlSWL1 associated with Phytophthora downyensis of litchi, characterized in that: Its amino acid sequence is shown in SEQ ID NO.2, or a similar sequence as shown in SEQ ID NO.2, which still has the same or similar function but is obtained by substitution, insertion or deletion of one or more amino acids.

2. The biomaterial related to the PlSWL1 pathogenic protein 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 plSWL1 protein, which is associated with the pathogenicity of Phytophthora licheeis; 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 nucleotide sequence of the nucleic acid molecule described in 1) is as shown in SEQ ID NO:1, or a similar sequence as shown in SEQ ID NO:1 that still has the same or similar function obtained by base insertion, deletion or substitution.

4. The biomaterial related to the pathogenic protein PlSWL1 of Phytophthora licheeis as described in claim 1, characterized in that: It can be any one of the following biological materials: 5) Nucleic acid molecules that inhibit or block the gene expression of the litchi downy mildew-associated protein PlSWL1; 6) A 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 PlSWL1; 7) The gene-deficient Phytophthora litchii pathogenicity-related protein PlSWL1 was prepared using the gene knockout vector described in 6).

5. The biomaterial according to claim 4, characterized in that: The nucleic acid molecule mentioned in 5) is the antisense RNA, siRNA, shRNA or sgRNA of the PlSWL1 gene.

6. The biomaterial according to claim 5, characterized in that: The sgRNA described is shown in any of the following sequences: PlSWL1-sgRNA1: 5'-CGCTGTCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCACAGCGATTGAGCCCAGTAC-3'; PlSWL1-sgRNA2: 5'-CACAATCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCATTGTGGTGCCGAATATTGT-3'.

7. The application of the litchi downy mildew pathogenicity-associated protein PlSWL1 as described in claim 1 or the biomaterial described in any one of claims 2-6, characterized in that: For any one or more of the following applications: i) Application in regulating the pathogenicity of Phytophthora downy mildew on litchi; ii) Application in regulating the expression levels of genes related to downy mildew in litchi; iii) Application in regulating the formation of oospores of Phytophthora downyensis on litchi; iv) Application in the prevention and control of lychee downy mildew; v) Application as a target in the design and screening of drugs against downy mildew of litchi.

8. A method for preventing and controlling litchi downy mildew caused by Phytophthora licheniformis, characterized in that: This is achieved by inhibiting or blocking the gene expression of the lychee downy mildew-associated protein PlSWL1 as described in claim 1.

9. A drug screening model for resistance to Phytophthora licheniformis, characterized in that: The drug screening model is a gene-deficient *Phytophthora litchifolia* strain of the pathogenic protein PlSWL1 described in claim 1.

10. The method for constructing the drug screening model against Phytophthora litchii as described in claim 9, characterized in that: Includes the following steps: (1) Design sgRNA based on the PlSWL1 gene sequence, and ligate the sgRNA with the pYF2.3G-Ribo-sgRNA vector to obtain the PlSWL1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlSWL1; And / or, based on the 1kb upstream and downstream sequences of the PlSWL1 gene sequence, primers for amplifying the left and right homologous arms were designed. Using Phytophthora litchii genomic DNA and NPTII synthetic fragments as templates, the left and right homologous arms and NPTII were amplified and ligated with the pBSSK vector to obtain the PlSWL1 gene knockout plasmid pBSSK::NPTII. (2) The PlSWL1 gene knockout plasmid pYF2.3G-Ribo-sgRNA::PlSWL1 and / or pBSSK::NPTⅡ were introduced into the protoplasts of wild-type strain of Phytophthora lichei. After screening and verification, the PlSWL1 gene knockout mutant was obtained, which is the drug screening model of Phytophthora lichei. The sgRNA mentioned in step (1) can be any of the following sequences: PlSWL1-sgRNA1: 5'-CGCTGTCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCACAGCGATTGAGCCCAGTAC-3'; PlSWL1-sgRNA2: 5'-CACAATCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCATTGTGGTGCCGAATATTGT-3'.

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