Myosin containing fyve domain and its coding gene and application

Abstract

The application discloses a myosin containing a FYVE domain from Phytophthora sojae and a coding gene and application thereof. The myosin containing a FYVE domain PsMyosinF of the application has an amino acid sequence shown in SEQ ID No. 4, and a coding gene thereof has a nucleotide sequence shown in SEQ ID No. 3. The main role of the PsMyosinF protein of the application is to be able to control the polar growth of the mycelium of Phytophthora sojae and sporangium formation. The gene provided by the application has a high application value in controlling the root rot of Phytophthora sojae, and a fungicide developed by taking the protein as a target has important practical significance in controlling the occurrence and prevalence of the root rot of Phytophthora sojae.

Description

Technical Field

[0001] This invention relates to a novel myosin from pathogenic oomycetes, its encoding gene and its applications, and particularly to the myosin PsMyosinF containing FYVE from Phytophthora soybean, its encoding gene and its applications. Background Technology

[0002] Phytophthora sojae is an important soil-borne plant pathogenic oomycete, first known to the world in North America in the 1950s. It has a very narrow host range, infecting only soybeans. Infection begins at the root and progresses to the stem. It can grow under moist conditions and in dense or heavily clay soils, causing seed rot, seedling lodging, and root and stem wilting and decay, leading to severe economic losses in the field.

[0003] Phytophthora soybeanis is a semi-parasitic oomycete with a well-developed, aspheric mycelium. Phytophthora soybeanis primarily invades the host's epidermal cells directly via infective hyphae, spreading between cells through polar growth. Under suitable conditions, the hyphae extend beyond the host epidermis, forming sporangia at their tips, which then release zoospores. These zoospores infect the host roots by releasing compounds towards them, or spread to the upper parts of the plant via splash, thus creating a reinfection process and leading to large-scale disease outbreaks.

[0004] In summary, the polar growth of the pathogenic hyphae and the formation of sporangia are essential processes for *Phytophthora sojae* to infect the host. If the polar growth of the hyphae and the formation of sporangia can be blocked, reinfection in the *Phytophthora sojae* disease cycle can be controlled, thus preventing large-scale outbreaks of the disease. Summary of the Invention

[0005] The inventors' research revealed that myosin containing the FYVE domain in Phytophthora soda is related to the polar growth of mycelia, sporangium formation, and pathogenicity of Phytophthora soda. Therefore, by controlling myosin containing the FYVE domain, the polar growth of mycelia and sporangium formation can be blocked, and the pathogenicity of Phytophthora soda can be reduced, thereby controlling (inhibiting or blocking) the large-scale spread and epidemic of Phytophthora soda.

[0006] Therefore, the present invention provides a myosin containing FYVE from Phytophthora sojae, named PsMyosinF, whose amino acid sequence is more than 93% similar to the amino acid sequence shown in SEQ ID No. 4, preferably more than 95%, more preferably more than 98%, and has the same function as the amino acid sequence shown in SEQ ID No. 4.

[0007] Generally speaking, the same species contains homologous and functionally identical or similar amino acid sequences (or protein sequences) or nucleic acid sequences, especially essential amino acid sequences (essential protein sequences) or essential nucleic acid sequences. These sequences are not necessarily 100% identical because different strains of the same species are susceptible to various mutations, including nonsense mutations, due to geographical conditions, external environmental factors such as temperature, humidity, and cultivars. However, because of the importance of their function to the survival or growth of the species, these mutations will not, or minimally, affect their function. Therefore, even within the same species, differences in amino acid sequences, such as those shown in SEQ ID No. 4, with the myosin of this invention are permissible within a certain range.

[0008] The amino acid sequence of the present invention may be an amino acid sequence as shown in SEQ ID No. 4, which has undergone substitution and / or deletion and / or addition of one or more amino acid residues and has the same function as the amino acid sequence as shown in SEQ ID No. 4.

[0009] For ease of purification, a fusion protein can also be obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 4; the tag can be a Poly-Arg (RRRRR), Poly-His (HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), c-myc (EQKLISEEDL), etc.

[0010] The myosin of this invention is generally derived from Phytophthora in soybean in nature, that is, it is a natural product, but it can also be expressed or synthesized artificially.

[0011] In this invention, the amino acid sequence of the myosin can specifically be as shown in SEQ ID No. 4. The protein shown in SEQ ID No. 4 consists of 1410 amino acid residues, wherein the sequence of the FYVE domain is the sequence of amino acid residues from the N-terminus of SEQ ID No. 4, from amino acid residues 1128 to 1181.

[0012] The second aspect of this invention provides the encoding gene PsMyosinF for the aforementioned myosin. The encoding gene preferably has the DNA sequence shown in SEQ ID No. 3, or a DNA sequence with at least 75% similarity to the DNA sequence shown in SEQ ID No. 3, more preferably at least 85%, and even more preferably at least 95%, and having the same function as the DNA sequence shown in SEQ ID No. 3, such as cDNA or recombinant DNA. For example, in this invention, the myosin DNA sequence includes the myosin DNA sequences present in different strains of Phytophthora soybeanis (e.g., Phytophthora soybeanis strain P6497).

[0013] In this invention, the DNA sequence of myosin (encoding gene and cDNA) can be specifically shown as SEQ ID No. 3.

[0014] The third aspect of this invention provides an RNA sequence transcribed from any of the above-mentioned DNA sequences, such as mRNA. Preferably, the RNA sequence has a similarity of 75% or more, more preferably 85% or more, and more preferably 95% or more, to the RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 3, and has the same function as the RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 3. Most preferably, the RNA sequence is an RNA sequence transcribed from the DNA sequence shown in SEQ ID No. 3.

[0015] The fourth invention provides biological materials related to the aforementioned myosin, encoding gene, or RNA molecule, which are any one of the following D1) to D10):

[0016] D1) An expression cassette containing the encoded gene;

[0017] D2) A recombinant vector containing the coding gene, or a recombinant vector containing the expression cassette described in D1);

[0018] D3) Recombinant microorganisms containing the coding gene, or recombinant microorganisms containing the expression cassette of D1), or recombinant microorganisms containing the recombinant vector of D2);

[0019] D4) A transgenic plant cell line containing the coding gene, or a transgenic plant cell line containing the expression cassette described in D1);

[0020] D5) Transgenic plant tissue containing the coding gene, or transgenic plant tissue containing the expression cassette described in D2);

[0021] D6) A transgenic plant organ containing the coding gene, or a transgenic plant organ containing the expression cassette described in D2);

[0022] D7) Nucleic acid molecules that inhibit the expression of the coding gene; preferably, the nucleic acid molecule is a nucleic acid molecule that knocks out the coding gene, or a nucleic acid molecule that silences the coding gene; wherein, the nucleic acid molecule that knocks out the coding gene includes sequences such as CCGUGUUGGUGUAGAUCUUGCUG, CCGGGACUCUUGCCGGCCACGGA and / or

[0023] The RNA molecule shown as UGCUCUCCGUCACCUCUCCGUGG, or the DNA molecule encoding the RNA molecule shown as SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. 32 in the sequence listing;

[0024] D8) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing or expressing the nucleic acid molecules described in D7);

[0025] D9) Nucleic acid molecules that inhibit the translation of the RNA molecule;

[0026] D10) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing or expressing the nucleic acid molecules described in D9).

[0027] The fifth objective of this invention is to provide the application of the aforementioned myosin, its encoding gene, or the aforementioned RNA molecule or the aforementioned biological material in controlling (inhibiting or blocking) the polar growth of oomycete hyphae and / or sporangium formation and / or reducing the pathogenicity of oomycetes;

[0028] Preferably, the oomycete is Phytophthora sojae, and most preferably Phytophthora sojae P6497.

[0029] The application of the myosin or its encoding gene as a target for screening pesticide formulations that control the polar growth of oomycete hyphae and / or control sporangium production and / or reduce the pathogenicity of oomycetes is also within the scope of protection of this invention.

[0030] The sixth objective of this invention is to provide a method for reducing the pathogenicity of oomycetes, the method comprising obtaining oomycetes with reduced pathogenicity by gene knockout, or inhibition of RNA sequence translation, or inhibition and / or inhibition of myosin activity; wherein the amino acid sequence of the myosin containing the FYVE domain is the amino acid sequence shown in SEQ ID No. 4; the DNA sequence is a DNA sequence capable of encoding the myosin; the RNA is an RNA sequence transcribed from the DNA sequence; wherein the oomycete is preferably Phytophthora sojae.

[0031] Preferably, the cDNA sequence of the myosin is the DNA sequence shown in SEQ ID No. 3.

[0032] The present invention also provides a method for controlling (inhibiting or blocking) the polar growth of oomycete hyphae or sporangium formation, comprising controlling (inhibiting or blocking) the polar growth of oomycete hyphae or sporangium formation by inhibiting the expression of the encoding gene of the aforementioned myosin or knocking out the encoding gene, or inhibiting the translation of the aforementioned RNA, or inhibiting and / or inactivating the activity of the aforementioned myosin; wherein the oomycete is *Phytophthora sojae*. The oomycete is preferably *Phytophthora sojae* strain P6497.

[0033] The present invention also provides a method for reducing the ability of oomycetes to infect a host, which reduces the ability of oomycetes to infect a host by controlling the polar growth of hyphae or the formation of sporangia as described in the claims; the host is soybean.

[0034] The above-described method for controlling the polar growth of *Phytophthora sojae* hyphae and sporangium formation involves knocking out any of the aforementioned DNA sequences to control these phenomena. The *Phytophthora sojae* strain includes *Phytophthora sojae* P6497.

[0035] In one embodiment of the present invention, the gene knockout method described above employs a CRISPR / Cas9-based gene knockout method.

[0036] Specifically, the CRISPR / Cas9-based gene knockout method involves transfecting the Donor vector of the target gene, sgRNA, and Cas9 co-expression plasmid into Phytophthora soybean and screening to obtain recombinant bacteria with inactivated target knockout proteins.

[0037] The Donor vector is a recombinant vector containing a sequence of 800-1500 bp upstream of the target gene to be knocked out, a selection gene, and a sequence of 800-1500 bp downstream of the target gene to be knocked out, connected sequentially.

[0038] Preferably, the Donor vector is a recombinant vector containing a 1000bp sequence upstream of the target gene to be knocked out, a selection gene, and a 1000bp sequence downstream of the target gene to be knocked out, connected sequentially. The selection gene is the GFP gene, the 1000bp sequence upstream of the target gene has the DNA sequence shown in SEQ ID No. 33, and the 1000bp sequence downstream of the target gene has the DNA sequence shown in SEQ ID No. 34.

[0039] The sgRNA sequences expressed by the sgRNA and Cas9 co-expression plasmids are CCGUGUUGGUGUAGAUCUUGCUG, CCGGGACUCUUGCCGGCCACGGA, and UGCUCUCCGUCACCUCUCCGUGG, respectively.

[0040] Preferably, the sgRNA and Cas9 co-expression plasmid is a CRISPR-Cas9 system expression vector containing a DNA fragment encoding sgRNA that targets the gene to be knocked out, wherein the DNA encoding sgRNA targeting the PsMyosinF gene is the DNA shown in SEQ ID No. 30, SEQ ID No. 31 or SEQ ID No. 32 in the sequence listing.

[0041] Preferably, the sgRNA and Cas9 co-expression plasmid is a starting vector in which the DNA fragments shown in SEQ ID No. 30, SEQ ID No. 31 or SEQ ID No. 32 are inserted between the BsaI and NneI enzyme recognition sites of PYF515, respectively, to obtain a recombinant expression vector expressing sgRNA and Cas9.

[0042] The seventh invention provides a method for detecting the transfection level of myosin containing the FYVE domain in *Phytophthora sojae*, comprising selecting any 80-300 bp, particularly 150-200 bp, target sequence from any of the DNA sequences mentioned above for reverse transcription quantitative PCR detection; preferably, it also includes an internal control sequence. The *Phytophthora sojae* strain includes *Phytophthora sojae* P6497.

[0043] This invention provides primer sequences for amplifying the expression level of myosin containing the FYVE domain in *Phytophthora sojae*, i.e., primer sequences for amplifying the target sequence. Preferably, the primer sequences are shown in SEQ ID No. 5 and SEQ ID No. 6; more preferably, the primer sequences for the internal reference sequence are shown in SEQ ID Nos. 13-14. The *Phytophthora sojae* includes strain P6497.

[0044] A primer and / or primer pair that amplifies the full-length DNA sequence encoding the myosin as described above or any segment thereof is also within the scope of protection of this invention.

[0045] Experiments have shown that the myosin provided in this invention plays a role in the growth and development of *Phytophthora sojae*. The knockout transformants obtained using PEG-CaCl2-mediated protoplast transformation exhibit slow hyphal growth and do not produce sporangia (the hyphal growth and sporangia formation of *Phytophthora sojae* P6497 and the knockout mutant ΔPsMyosinF are shown in the figure). Figure 4 As shown), the phenotype of the full-length transformant was restored after restoration (the mycelial growth and sporangium formation of the restored transformant were as shown in the figure). Figure 4 (As shown). Therefore, the myosin PsMyosinF containing the FYVE domain in Phytophthora soysarum can regulate the polar growth of hyphae and the formation of sporangia. Inhibiting the function of this protein can control reinfection in the Phytophthora soysarum disease cycle and control large-scale epidemics. This invention provides a technical basis for further research on the development process and molecular detection technology of Phytophthora soysarum, as well as the prevention and control of plant diseases caused by Phytophthora soysarum. Attached Figure Description

[0046] Figure 1 This is a graph showing the expression pattern of the PsMyosinF gene in Phytophthora soybeanis at different developmental stages (the horizontal axis from left to right represents: hyphae (My), sporangia (Sp), zoospores (Zo), resting spores (Cy), resting spore germination (Cg), and infection of soybean leaves at 1.5h, 3h, 6h, 12h, 24h, and 48h).

[0047] Figure 2 The diagram shows a knockout vector, with the left side showing a homologous arm replacement vector and the right side showing an sgRNA vector.

[0048] Figure 3 PCR identification results for the PsMyosinF knockout mutant of Phytophthora soybean (left) and gene expression analysis for the full-length and truncated complement transformants (right). PCR electrophoresis results from left to right: wild-type Phytophthora soybean strain (P6497), knockout mutants (ΔPsMyosinF-1, ΔPsMyosinF-2, and ΔPsMyosinF-3), water, and control strain (CK).

[0049] Figure 4 Images show the mycelial growth, colony morphology, and sporangium morphology of wild-type Phytophthora soybean strain P6497, the PsMyosinF gene knockout mutant ΔPsMyosinF, and the complemented transformant.

[0050] Figure 5Images show soybean leaves infected with mycelium cakes 48 hours after inoculation and the pathogenicity assay of the knockout mutants in soybean leaves. From left to right, the images show wild-type Phytophthora soybeanis strain P6497, PsMyosinF knockout transformants (ΔPsMyosinF-1, ΔPsMyosinF-2, and ΔPsMyosinF-3), and complement transformants infected with mycelium cakes 48 hours after inoculation. Detailed Implementation

[0051] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] The wild-type strain of *Phytophthora sojae*, P6497, was a standard strain of *Phytophthora sojae* donated by Professor Brett M. Tyler of the United States. The above strains are only materials used in the embodiments of this invention; in fact, *Phytophthora* strains can be obtained through any commercially available means when applying the screening markers described in this invention.

[0053] All of the above strains have been identified using existing morphological and molecular biological methods.

[0054] Example 1: Cloning of the PsMyosinF gene containing the FYVE domain from Phytophthora soybeanis

[0055] 1.1 Total RNA extraction from Phytophthora soybean

[0056] After incubating *Phytophthora sojae* strain P6497 on 10% V8 solid medium in the dark at 25°C for 4 days, 5 mm diameter mycelial cakes were collected from the colony edge and inoculated into 10% V8 liquid medium. After incubation in the dark at 25°C for 4 days, mycelia were harvested. Approximately 30 mg of mycelia were transferred to a 2 mL sterile, mold-free centrifuge tube, along with two 5 mm diameter steel balls. The tube was then frozen in liquid nitrogen and ground into powder using a ball mill. RNA was extracted from *Phytophthora sojae* using the Promega SV Total RNA Extraction Kit (Z3100), following the instructions for plant tissue RNA extraction in the kit.

[0057] 1.2 Synthesis of first-strand cDNA by reverse transcription from Phytophthora soybeanis

[0058] First-strand cDNA synthesis was performed using Takara Bio's technology. The RT reagent kit with gDNAEraser (Perfect Real Time) kit (RR047A) was used. The specific steps are as follows:

[0059] (1) Removal of genomic DNA (10 μL reaction system): 2 μL of 5×gDNA Eraser Buffer; 1 μL of gDNA Eraser; 2 μL of total RNA; 5 μL of RNase-free ultrapure water.

[0060] (2) Reverse transcription reaction (10 μL reaction system): 10 μL of the reaction solution from step (1); 5× Buffer 4μL; Enzyme Mix 1 μL; RT Primer Mix 1 μL; RNase-free ultrapure water 4 μL.

[0061] Reaction conditions: 37℃ for 15 min; 85℃ for 5 s; store at 4℃. The obtained cDNA was diluted 4-fold for Real-time PCR.

[0062] 1.3 Cloning of the PsMyosinF gene

[0063] Primers for cloning the myosin gene containing the FYVE domain from *Phytophthora sojae* were designed: PsMyosinF-F: ATGGACGTGGGCGCCGAG (SEQ ID No. 1); PsMyosinF-R: TCATAGAAATTCGCTATCGCTATC (SEQ ID No. 2). Using the *Phytophthora sojae* cDNA and genome obtained in step 1.2 as templates, PCR amplification was performed using the above primers. The amplified products were recovered and ligated into T1-simple vectors (Beijing TransGen Biotech Co., Ltd., Beijing), and transformed into *E. coli* T1 competent cells. Positive clones were screened, and plasmids from the positive clones were extracted and sequenced. Sequencing results showed that the PsMyosinF gene cDNA has the nucleotide sequence shown in SEQ ID No. 3. The inventors confirmed through genome amplification and sequencing that the encoding gene is consistent with the cDNA sequence. SEQ ID No. 3 consists of 4233 nucleotides, encoding a protein of 1410 amino acids (sequence shown in SEQ ID No. 4). This protein was named PsMyosinF. The FYVE domain consists of amino acid residues from amino terminus 1128 to 1181 of SEQ ID No. 4.

[0064] SEQ ID No. 3:

[0065]

[0066]

[0067]

[0068] SEQ ID No. 3:

[0069]

[0070]

[0071] Example 2: Expression pattern analysis of PsMyosinF gene at different developmental stages of Phytophthora soybeanis

[0072] Selected reagent kit: Takara TBGreen TM Premix Ex Taq TM II (Tli RNaseH Plus) (Code: RR820A). Diluted mycelial stage cDNA samples at 0, 4, and 4% concentrations respectively. 2 4 3 4 4 4 5 4 6 Using PsActin (primer sequences see SEQ ID No. 13, SEQ ID No. 14) as the internal reference gene and PsMyosinF as the detection gene, a standard curve for real-time quantitative PCR was established. The gene primer amplification efficiency needed to be above 90%, and appropriate sample dilution concentrations were selected for the next step of the experiment.

[0073] Samples at different developmental and infection stages, diluted 4-fold, were used as templates. PsActin was used as an internal reference gene. Real-time quantitative PCR was performed using the detection primers PsMyosinF-qPCR-F / PsMyosinF-qPCR-R (sequences shown in SEQ ID No. 5 and SEQ ID No. 6). The qPCR reaction system consisted of 20 μL: 10 μL TB Green Premix Ex Taq II, 1.6 μL template, 0.8 μL each of F / R primers, and 6.8 μL ddH2O. The reaction program was a two-step method: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. Through 2... -ΔΔCt Calculate the relative expression level of the PsMyosinF gene at different developmental stages of Phytophthora soybean.

[0074] The results are as follows Figure 1 According to the above, PsMyosinF was significantly highly expressed in the zoospore stage (zo) and the resting spore germination stage (cg) of Phytophthora soybeani P6497 mycelial stage (my) after normalization.

[0075] Example 3: Obtaining knockout transformants of the PsMyosinF gene from soybean Phytophthora infestans

[0076] 3.1 Construction of a knockout vector for the PsMyosinF gene of Phytophthora soybean

[0077] (1) Obtaining candidate sgRNAs

[0078] The sgRNA for PsMyosinF was designed using EuPaGDT (http: / / grna.ctegd.uga.edu / ), by entering the full genome length into the website for searching.

[0079] (2) Secondary structure screening

[0080] The secondary structures of candidate sgRNAs were analyzed using a web tool (http: / / rna.urmc.rochester.edu / RNAstructureWeb / Servers / Predict1 / Predict1.html), and candidate sgRNAs were selected based on the number of hairpin complementary bridges that are less than or equal to 3.

[0081] (3) Off-target analysis

[0082] Off-target analysis of sgRNA was performed using FungiDB (www.fungidb.org). The input consisted of 23 bases, including the PAM region, and was compared. Cases with 9 to 23 (including NGG) complete matches were considered to have a high risk of off-target effects.

[0083] (4) Select an ideal sgRNA for synthesis (the DNA sequences expressing sgRNA are shown in SEQ ID No. 30, SEQ ID No. 31 and SEQ ID No. 32, and the sgRNA sequences are CCGUGUUGGUGUAGAUCUUGCUG, CCGGGACUCUUGCCGGCCACGGA, and UGCUCUCCGUCACCUCUCCGUGG, respectively. Target the 5' end nucleotide sequences 291-313, 622-644, and 948-970 of SEQ ID No. 3, respectively.

[0084] (5) Using PYF515 (published vector, Fang Y, Cui L, Gu B, et al. 2017. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR / Cas9. Current Protocols in Microbiology, 44:21A.1.1-21A.1.26.) as the backbone vector, an sgRNA expression vector (structure shown in the original text) was constructed according to the published method (described in Fang Y, Cui L, Gu B, et al. 2017. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR / Cas9. Current Protocols in Microbiology, 44:21A.1.1-21A.1.26.). Figure 2 (sgRNA vector). Annealing of sgRNA-encoded DNA (30 μL system): 3 μL sense strand, 3 μL antisense strand, 3 μL 10×T4 DNA Ligase Buffer (NEB), 2 μL T4 Polynudeotide and 19 μL ddH2O, 37℃, 30 min; add 4 μL 0.5M NaCl, 100℃, 2 min; slowly cool at room temperature for about 3-4 h; dilute 1 μL, add 499 μL ddH2O; digest PYF515 plasmid with BsaI and NneI restriction enzymes to linearize the plasmid; ligation of plasmid: 3 μL diluted and annealed sgRNA fragment-encoded DNA (sequence see SEQ ID No. 30, SEQ ID No. 31 or SEQ ID No. 32), 2 μL 10×T4 DNA Ligase Buffer (NEB), 1 μL T4...

[0085] DNA polynudeotide, 50 ng PYF515 linear plasmid, ddH2O added to 20 μL, incubated at 25°C for 30 min. The ligation product was then transformed into E. coli DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 35 s, then on ice for 2 min. Antibiotic-free LB agar was added and the cells were shaken for 60 min. The bacterial culture was then plated onto LB agar plates containing Amp antibiotics and incubated overnight at 37°C. The clones were then amplified and sequenced using universal primers M13F (sequence: TGTAAAACGACGGCCAGT) / RPL41-F (sequence: CAAGCCTCACTTTCTGCTGACTG) to verify the clones. The recombinant vector containing the fragment shown in SEQ ID No. 30, which was verified to be correct, was named PYF515-sgRNA1. This vector expresses the nucleic acid sequence targeting the 5' end of SEQ ID No. 3, positions 291-313. The recombinant vector containing the fragment shown in SEQ ID No. 31, which was verified to be correct, was named PYF515-sgRNA2. This vector expresses the nucleic acid sequence targeting the 5' end of SEQ ID No. 3, positions 622-644. The recombinant vector containing the fragment shown in SEQ ID No. 32, which was verified to be correct, was named PYF515-sgRNA3. This vector expresses the nucleic acid sequence targeting the 5' end of SEQ ID No. 3, positions 948-970.

[0086] (6) Using the genomic DNA of *Phytophthora sojae* P6497 as a template, primer pairs were used to amplify the upstream 1000bp fragment (sequence in SEQ ID No. 33) and downstream 1000bp fragment (sequence in SEQ ID No. 34) of the target gene (sequences of the primer pairs for the upstream 1000bp fragment are shown in SEQ ID No. 7 and SEQ ID No. 8, and the sequence of the primer pairs for the downstream 1000bp fragment are shown in SEQ ID No. 11 and SEQ ID No. 12). Using PYF515 as a template, the GFP fragment was amplified using primer pairs (sequences in SEQ ID No. 9 and SEQ ID No. 10). pBluescript II SK was used to amplify the fragment. + As the backbone vector, pBluescript II SK was digested with EcoRI and BamHI (enzymes purchased from NEB) as restriction sites. + The plasmid was used to construct a homologous arm substitution vector (structure shown in Figure 1) using an in-fusion kit (Takara Code No. 639650). Figure 2 (Donor carrier). The HD Cloning Kit fuses and ligates three amplified fragments into the cloning vector pBluescript II SK. +After digestion with EcoRI and BamHI, the ligation product was transferred into E. coli DH5α competent cells at 50℃ for 15 min. The cells were then incubated on ice for 30 min, heat-shocked at 42℃ for 35 s, and then incubated on ice for 2 min. LB agar without antibiotics was added and the cells were shaken for 60 min. The bacterial culture was then plated on LB agar plates containing Amp antibiotics and incubated overnight at 37℃. The clone was then amplified and sequenced using universal primers M13F (sequence: TGTAAAACGACGGCCAGT) / M13R (sequence: CAGGAAACAGCTATGACC). The verified recombinant expression vector containing the sequentially linked upstream 1000 bp sequence of PsMyosinF, the GFP gene sequence, and the downstream 1000 bp sequence of PsMyosinF was named pBS-GFP-PsMyosinF.

[0087] 3.2 Obtaining knockout transformants of the PsMyosinF gene from Phytophthora soybean

[0088] PsMyosinF gene knockout transformants were obtained using CaCl2-PEG mediated protoplast transformation (described in Fang and Tyler, 2016. Efficient disruption and replacement of an effector gene in the oomycete Phytophthora sojae using CRISPR / Cas9). Specifically, the sgRNA expression vectors constructed in section 3.1 (PYF515-sgRNA1, PYF515-sgRNA2, and PYF515-sgRNA3) were transformed into three groups simultaneously: the first group consisted of PYF515-sgRNA1 and PYF515-sgRNA2; the second group consisted of PYF515-sgRNA2 and PYF515-sgRNA3; and the third group consisted of PYF515-sgRNA1 and PYF515-sgRNA3) and the homologous arm replacement vector pBS-GFP-PsMyosinF. The transformed individuals were then transformed into *Phytophthora sojae* P6497 culture medium. The transformed individuals were screened by incubating them on G418 resistant V8 plates at 25°C. Mycelial blocks were then taken from the edge of the colony and inoculated onto V8 plates lined with cellophane. After incubation in the dark at 25°C for 6 days, the mycelia were scraped off, and DNA was extracted from the transformed individual samples.

[0089] Three primer pairs were used to verify the knockout transformant. Primer pairs PsMyosinF-QCYZ-F1 / R1 (see sequences 15 and 16 in the sequence listing) and PsMyosinF-QCYZ-F2 / R2 (see sequences 19 and 20 in the sequence listing) were used to verify whether gene substitution had occurred, and PsMyosinF-QCYZ-F3 / R3 (see sequences 17 and 18 in the sequence listing) were used to verify the internal structure of the gene. If PsMyosinF-QCYZ-F1 / R1 and PsMyosinF-QCYZ-F2 / R2 showed bands when PCR amplified with the suspected transformant, and PsMyosinF-QCYZ-F3 / R3 showed no band when PCR amplified with the suspected transformant, then the suspected transformant could be identified as a positive homozygous transformant; if all three primer pairs showed bands, then the suspected transformant could be identified as a positive heterozygous transformant. Figure 3 As shown, a total of three homozygous PsMyosinF knockout transformants were obtained: ΔPsMyosinF-1, ΔPsMyosinF-2 and ΔPsMyosinF-3.

[0090] Example 4: Obtaining complement transformants from overexpression of the PsMyosinF gene in soybean Phytophthora infestans

[0091] Using the cDNA of Phytophthora sojae in soybean as a template, PCR amplification was performed using primers (sequences shown in SEQ ID No. 21 and SEQ ID No. 22). The amplified PsMyosinF gene cDNA fragment product was recovered and digested with ApaI and SpeI (enzymes purchased from NEB). At the same time, the PYF3 plasmid (published vector, Fang Y, Cui L, Gu B, et al. 2017. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR / Cas9. Current Protocols in Microbiology, 44:21A.1.1-21A.1.26.) was amplified for enzyme digestion. The digested PsMyosinF gene was ligated into the digested PYF3 plasmid. The ligation system was as follows (T4 ligase was purchased from Takara): 10 μL total volume (reaction conditions: 25℃ for 30 min), including 1 μL of the digested PYF3 plasmid, 5 μL of the digested PsMyosinF gene fragment, 2 μL of 5×T4 DNA Ligase Buffer, 0.5 μL of T4 DNA Ligase, and 1.5 μL of ddH2O. Sequencing yielded the full-length overexpression vector of PsMyosinF. Using the full-length overexpression vector as a template, amplification of truncated fragments of the key domain was performed (SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, and SEQ ID No. 24 were used to amplify the truncated Motor domain fragment of the constructed key domain truncated overexpression vector, with a total amplification product of 2070 nucleotides; sequences SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 25, and SEQ ID No. 26 were used to amplify the truncated FYVE domain fragment of the constructed key domain truncated overexpression vector, with a total amplification product of 4071 nucleotides; sequences SEQ ID No. 21 and SEQ ID No. 27 were used to amplify the truncated GAF ​​domain fragment of the constructed key domain truncated overexpression vector, with a total amplification product of 3723 nucleotides). The HD Cloning Kit (Takara Code No. 639650) was used to construct a truncated overexpression vector for the key domain of PsMyosinF. The amplified fragments corresponding to each truncated domain were fused and ligated into the cloning vector PYF3 (digested with SpeI and ApaI). After incubation at 50°C for 15 min, the ligation product was transformed into *E. coli* DH5α competent cells. The cells were incubated on ice for 30 min, heat-shocked at 42°C for 35 s, and then on ice for 2 min. After adding antibiotic-free LB agar and shaking for 60 min, the bacterial culture was plated on LB agar plates containing Amp antibiotics and incubated overnight at 37°C. The clones were then amplified and sequenced using primers pTOR-F (sequence: CCAAGTCCCAACCGACTCTT) / pTOR-R (sequence: GTTCTACAAACGGCCTTCTT) to verify the clones. The verified recombinant expression vectors were named PYF3-PsMyosinF-ΔMotor, PYF3-PsMyosinF-ΔFYVE, and PYF3-PsMyosinF-ΔGAF, respectively.

[0092] Using the PsMyosinF knockout transformant ΔPsMyosinF-1 obtained in step 3.2 as experimental material, protoplast transformation was performed using CaCl2-PEG mediated transformation. Overexpression complementation transformants of full-length and truncated PsMyosinF were verified using primer pairs (sequences shown in SEQ ID No. 28-29). The correctly verified full-length PsMyosinF complementation transformant was named ΔPsMyosinF-C; the correctly verified complementation transformant of the truncated Motor domain fragment was also named ΔPsMyosinF-C. ΔMotor The complemented transform of the correctly verified truncated FYVE structural domain is named ΔPsMyosinF-C. ΔFYVE The complement of the correctly verified truncated GAF ​​domain is named ΔPsMyosinF-C. ΔGAF .

[0093] Example 5: Biological trait analysis of knockout and complement transformants of the PsMyosinF gene from soybean Phytophthora infestans.

[0094] (1) Detection of mycelial growth rate

[0095] The knockout transformant ΔPsMyosinF, the full-length complement transformant ΔPsMyosinF-C, and the complement transformant with truncated structural domains (ΔPsMyosinF-C) of the PsMyosinF gene from the tested strain *Phytophthora spp.* were compared. ΔMotor ΔPsMyosinF-C ΔFYVE and ΔPsMyosinF-C ΔGAFAfter inoculating the colonies onto V8 plates and incubating in the dark at 25°C for 6 days, mycelial cakes were collected along the edge of the colonies using a 5mm diameter punch and inoculated onto V8 plates. The plates were then incubated in the dark at 25°C for 6 days. The colony diameter was measured using the cross-sectional method. The experiment was performed in triplicate. *Phytophthora soybeani* P6497 was used as a control.

[0096] like Figure 4 As shown, compared with the wild-type soybean Phytophthora strain P6497, the knockout mutant ΔPsMyosinF exhibited hyphal swelling and a significantly decreased growth rate. The full-length complemented transformant ΔPsMyosinF-C and the GAF domain-truncated complemented transformant ΔPsMyosinF-C also showed significant growth. ΔGAF The mycelial growth characteristics were restored, and the truncated complement transformant ΔPsMyosinF-C from the Motor domain was restored. ΔMotor The truncated complemented transformant ΔPsMyosinF-C of the FYVE structural domain ΔFYVE The mycelial growth characteristics did not recover.

[0097] (2) Determination of sporangia and zoospores

[0098] The knockout transformant ΔPsMyosinF, the full-length complement transformant ΔPsMyosinF-C, and the complement transformant with truncated structural domains (ΔPsMyosinF-C) of the PsMyosinF gene from the tested strain *Phytophthora spp.* were compared. ΔMotor ΔPsMyosinF-C ΔFYVE and ΔPsMyosinF-C ΔGAF After inoculating onto V8 plates and culturing in the dark at 25°C for 6 days, mycelial cakes were collected along the edge of the colony using a 5mm diameter punch. Ten mycelial cakes were placed in a petri dish containing 20mL of 10% V8 liquid medium, with the mycelial side facing upwards and the water level completely submerged. The mixture was incubated in the dark at 25°C for 2 days. The V8 liquid medium was then discarded, and the mycelial clusters were resuspended in 20mL of sterile water and rinsed five times, with 30min intervals between each rinse. After rinsing, 20mL of sterile water was added, and the mixture was incubated in the dark at 25°C for 8-12 hours to induce sporangium production. The number of sporangia produced per field of view for each strain was examined under a microscope. The experiment was performed in triplicate. *Phytophthora soybeani* P6497 was used as a control.

[0099] After microscopic examination, the sporangia were cultured in the dark at 25°C for 1-3 hours to induce the production of zoospores. The zoospore suspension was collected, vortexed for 30 seconds, and then examined under a microscope using a hemocytometer to calculate the zoospore yield. The experiment was performed in triplicate.

[0100] The results are as follows Figure 4As shown, under sporulation induction treatment, the wild-type *Phytophthora soybean* strain P6497 can form sporangia normally, while the knockout mutant ΔPsMyosinF does not produce sporangia. The full-length complemented transformant ΔPsMyosinF-C and the GAF domain truncated complemented transformant ΔPsMyosinF-C are also shown. ΔGAF It can produce sporangia normally, while the Motor domain truncated complemented transformant ΔPsMyosinF-C ΔMotor The truncated complemented transformant ΔPsMyosinF-C of the FYVE structural domain ΔFYVE It cannot produce sporangia.

[0101] (3) Pathogenicity of transformants in detached leaves

[0102] Pathogenicity of detached leaf mycelium: Knockout transformants (ΔPsMyosinF-1, ΔPsMyosinF-2, and ΔPsMyosinF-3) and full-length complemented transformant ΔPsMyosinF-C of *Phytophthora sojae* gene were cultured in the dark at 25°C for 6 days. Mycelium was then collected from the colony edges using a 5mm diameter punch. Two layers of absorbent paper were placed in a food storage container, and ten healthy, isometric, and age-matched soybean leaves (second pair of true leaves) were added. The collected mycelium was inoculated onto the soybean leaves, one mycelium per leaf, with at least 10 leaves inoculated per group. The inoculated soybean leaves were then placed in the dark at 25°C (RH = 60%-80%) for 3 days. The area of ​​lesions was measured, recorded, and photographed. The experiment was performed in triplicate. *Phytophthora sojae* P6497 was used as a control.

[0103] like Figure 5 As shown, when soybean detached leaves were inoculated with mycelium cake, the pathogenicity of the knockout mutant was significantly reduced compared with the wild-type soybean Phytophthora strain P6497, while the pathogenicity of the full-length complement transformant was restored.

Claims

1. The application of knocking out the myosin encoding gene containing the FYVE domain shown in SEQ ID No. 4 of Phytophthora soybeanis in inhibiting the polar growth of oomycete hyphae and / or inhibiting sporangium production and / or reducing the pathogenicity of oomycetes.

2. The application according to claim 1, characterized in that, The encoding gene for the myosin containing the FYVE domain is the DNA shown in SEQ ID No.

3.

3. A method for reducing the pathogenicity of *Phytophthora sojae*, comprising obtaining oomycetes with reduced pathogenicity by inhibiting or inactivating the expression of a myosin encoding a myosin containing an FYVE domain in oomycetes; wherein the amino acid sequence of the myosin containing the FYVE domain is the amino acid sequence shown in SEQ ID No. 4; and wherein the oomycete is *Phytophthora sojae* (… Phytophthora sojae ); The method for inhibiting or inactivating the expression of the gene encoding myosin containing the FYVE domain in oomycetes is to knock out the gene.

4. The method according to claim 3, characterized in that, The encoding gene for the myosin containing the FYVE domain is the DNA shown in SEQ ID No.

3.

5. A method for inhibiting or blocking the polar growth of oomycete hyphae or sporangium formation, comprising inhibiting or blocking the polar growth of oomycete hyphae or sporangium formation by inhibiting or inactivating the expression of a myosin encoding a myosin containing an FYVE domain in oomycetes; The amino acid sequence of the myosin containing the FYVE domain is the amino acid sequence shown in SEQ ID No. 4; the oomycete is Phytophthora soybeanis (… Phytophthora sojae ); The method for inhibiting or inactivating the expression of the gene encoding myosin containing the FYVE domain in oomycetes is to knock out the gene.

6. The method according to claim 5, characterized in that, The encoding gene for the myosin containing the FYVE domain is the DNA shown in SEQ ID No. 3.

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