Application of flagellum-associated protein PbFAP-102 in plasmodiophora brassicae and coding gene of flagellum-associated protein PbFAP-102 in prevention and control of rape clubroot

Abstract

The invention belongs to the field of biological agriculture, and relates to application of flagellum-associated protein PbFAP-102 in plasmodiophora brassicae and a coding gene thereof in prevention and control of rape clubroot. According to the research, flagellar associated protein (PbFAP-102) which is expressed at a high level in each stage is screened from a plasmodiophora brassicae ZJ-1 transcriptome, an RNAi carrier of the gene is constructed, a corresponding transgenic plant is obtained, and a disease resistance test of plasmodiophora brassicae shows that the two PbFAP-102-RNAi transgenic lines both show better disease resistance phenotypes, and the two PbFAP-102-RNAi transgenic lines show better disease resistance phenotypes. Therefore, the flagellum-related protein PbFAP-102 of plasmodiophora brassicae can be used as a gene resource for resisting clubroot.

Description

Technical Field

[0001] This invention belongs to the field of bio-agriculture and relates to the application of flagella-associated protein PbFAP-102 and its encoding gene in the control of clubroot disease in rapeseed. Background Technology

[0002] Clubroot disease, caused by Plasmodiophora brassicae, is a global soil-borne disease that poses a serious threat to the yield and quality of Brassica genus crops in the Brassicaceae family. It has become one of the most damaging diseases affecting Brassica plants. Currently, Plasmodiophora brassicae lacks a stable genetic system, and research on its pathogenic mechanism is relatively lagging. Furthermore, the dormant spores of Plasmodiophora brassicae can survive in the soil for a long time, making the control of clubroot disease particularly difficult. Therefore, finding genetic and germplasm resources resistant to clubroot is of great significance for protecting the safe production of Brassica genus crops. Host-induced gene silencing (HIGS) technology involves screening for disease-resistant genes, designing a hairpin-structured vector carrying a pathogen-specific gene fragment, and transforming it into host plants. Transgenic plants produce corresponding dsRNA and siRNA, which enter the pathogen during host-pathogen interactions, degrade pathogen mRNA, and silence target genes to protect the host plant from pathogen attack (Nunes and Dean 2012, Qi et al 2019, Koch et al 2021). Using HIGS technology, dsRNA targeting the thiamine transporter was introduced into cotton, successfully generating two stable transgenic lines. Compared to the wild type, *Verticillium dahliae* colonization and spread in the roots of RNAi cotton were significantly reduced, and the disease index was significantly decreased. Under field conditions, RNAi transgenic cotton also showed significantly enhanced disease resistance and yield (Wang et al 2024). Flagella, also called cilia, protruding from the surface of eukaryotic cells, are microtubule-based organelles widely distributed in protozoa and vertebrates, possessing functions of movement, sensing, and secretion. In liquids, the flagella of organisms drive the movement of the cell itself or the liquid through beating. Eukaryotic flagella are mainly composed of a flagellum and a basal body. The flagellum has a "9+2" shape, meaning it has two central microtubules (complete microtubules with 13 subunits surrounding them) and a ring of nine microtubule duodenal structures surrounding it. The A tube is a complete microtubule, while the B tube is composed of 10 subunits, with the other three subunits sharing the A tube. The A subfibril also has two dynein walls, which mainly participate in flagellar bending movement through Ca+-activated ATPase. Li Ziyin et al. discovered a signal transduction pathway regulating flagellar inheritance in protozoa (Trypanosoma brevicornu). This work not only elucidated the molecular mechanism by which the dephosphorylase KPP1 regulates flagellar inheritance but also laid the foundation for future drug design and development targeting KPP1 (An et al 2021).Further research has shown that there is a special microtubule protein modification in human sperm flagella—glycine modification—which is crucial for sperm to maintain a straight line of movement. When this modification is disturbed, sperm can only circle in place and cannot move in a straight line, which can lead to certain forms of male infertility, thus revealing a new mechanism of male infertility (Gadadhar et al. 2021). Summary of the Invention

[0003] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a flagellation-associated protein, PbFAP-102, from *Plasmodium*.

[0004] Another objective of this invention is to provide the gene encoding the flagella-associated protein PbFAP-102 in *Plasmodium* and its applications.

[0005] Another object of the present invention is to provide a method for cultivating plants resistant to clubroot pathogen.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The amino acid sequence of PbFAP-102, a flagellation-associated protein in *Plasmodium*, is shown in SEQ ID NO.2.

[0008] The CDS sequence of the gene encoding the flagella-associated protein PbFAP-102 in the aforementioned *Plasmodium* is shown in SEQ ID NO.1.

[0009] Application of substances that reduce the expression level of flagella-associated protein PbFAP-102 in the aforementioned clubroot bacteria in enhancing plant resistance to clubroot disease.

[0010] Preferably, the substance used to reduce the expression level of flagella-associated protein PbFAP-102 in the *Plasmodium* species of claim 1 is selected from host-induced gene silencing RNAi vectors targeting this gene, gene editing systems, siRNA, dsRNA, and ordinary RNAi vectors.

[0011] The application of negative regulation of the coding genes in enhancing plant resistance to clubroot.

[0012] A host-induced gene silencing RNAi vector for silencing the PbATG8 gene, wherein the host-induced gene silencing RNAi vector uses the CDS sequence fragment shown in SEQ ID NO.3 as an interfering sequence.

[0013] Engineered bacteria containing the aforementioned RNAi vector.

[0014] Any of the following applications of the RNAi vector and the engineered bacteria:

[0015] 1) Increase the plant's resistance to clubroot disease;

[0016] 2) Prepare products that enhance the resistance of plants to clubroot disease;

[0017] 3) Cultivate plants with improved resistance to clubroot disease;

[0018] 4) Prepare products that cultivate plants with enhanced resistance to clubroot disease;

[0019] 5) Improve plants with high resistance to clubroot disease or prepare products made from plants with high resistance to clubroot disease;

[0020] 7) Inhibits the growth and development of clubroot pathogens;

[0021] 8) Prepare products that inhibit the growth and development of clubroot bacteria.

[0022] Preferably, the plant is selected from rapeseed.

[0023] A method for cultivating clubroot-resistant plants includes introducing the host-induced gene-silencing RNAi vector into a target plant to obtain clubroot-resistant plants, wherein the clubroot-resistant plants exhibit higher resistance to clubroot than the target plant, and the RNAi vector targets the gene encoding the flagella-associated protein PbFAP-102 in the clubroot fungus.

[0024] Beneficial effects:

[0025] This study screened flagellar-associated protein (PbFAP-102) at high levels in all stages of the *Plasmodiophora* ZJ-1 transcriptome. An RNAi vector for this gene was constructed, and corresponding transgenic plants were obtained. *Plasmodiophora* disease resistance tests showed that both PbFAP-102-RNAi transgenic lines exhibited good disease resistance phenotypes, indicating that the flagellar-associated protein PbFAP-102 from *Plasmodiophora* can serve as a gene resource for resistance to clubroot disease. Attached Figure Description

[0026] Figure 1 PbFAP-102-RNAi fragment amplification, expression levels at different stages, and vector identification.

[0027] PbFAP-102-RNAi positive strand PCR amplification, M: DL 2000 Marker (A). PbFAP-102-RNAi antisense strand PCR amplification, M: DL 2000 Marker (B). PbFAP-102-RNAi expression level in the life cycle of *Plasmodium*, IN represents the cortical infection stage, PZ represents the zoospore stage, and RS represents the dormant spore stage (C). PbFAP-102-RNAi vector identification: 1: positive strand PCR detection; 2: antisense strand PCR detection; 3: detection by double digestion of plasmid with KpnI and SpeI; M: DL 2000 Marker (D).

[0028] Figure 2 HIGS screening of transgenic materials

[0029] DNA was extracted from 20 PbFAP-102-RNAi (A) T0 generation HIGS transgenic materials and identified using specific PCR primers. The results showed that 16 positive seedlings were obtained. (B) Phenotype of positive HIGS transgenic materials after 4 weeks of growth.

[0030] Figure 3 Phenotypic and disease index of PbFAP-102 transgenic plants inoculated with *Plasmodium*, and expression levels of the target gene and content of *Plasmodium* detected by qPCR.

[0031] Three weeks after inoculation of ZS11 and two different PbFAP-102 transgenic lines with *Plasmodium* (each line inoculated with 1 × 10⁻⁶ dormant spores), 7 Plant phenotype (A) and disease index (B, C) were analyzed using n=20-40 biological replicates. Total RNA (D) and DNA (E) were extracted from the roots of PbFAP-102-RNAi transgenic lines and wild-type Arabidopsis thaliana 3 weeks after inoculation with *P. t-test*. The content of *P. t-test* in diseased rapeseed roots was quantified by qPCR. The ordinate represents the fold increase in *P. t-test* content relative to ZS11 for different lines. The relative biomass of *P. t-test* was used to measure the expression levels of target genes in diseased rapeseed roots. The expression levels of target genes were measured using target-specific PCR primers, with the *P. t-test* as an internal control. n=3 biological replicates were used, and t-tests were employed to analyze differences (CE). Detailed Implementation

[0032] Example 1

[0033] Analysis of the *Plasmodiophora* genome data yielded 61 flagellation-related protein genes. Transcriptome data analysis and comparison with NCBI data identified 11 genes highly expressed during the cortical infection stage of *Plasmodiophora* growth and development. PbFAP-102 was one of these genes. Its high expression during cortical infection suggests that this gene may influence *Plasmodiophora* infection and thus its pathogenicity. Figure 1 A).

[0034] Example 2

[0035] 1. Experimental Methods

[0036] 1.1 Extraction of total DNA from *Plasmodium* (CTAB method)

[0037] Add dormant spores of *Cladophora mycoides* (collected from diseased rapeseed fields in Zhijiang City and identified as clubroot-infected root system (Williams 1 physiological race) ZJ-1) or diseased roots to a pre-cooled mortar. Grind thoroughly with liquid nitrogen. Transfer the powder to a 2 mL centrifuge tube, add 700 µL of 2% CTAB extraction buffer (preheated to 65°C, 0.2 g powder added to 1 mL buffer), quickly invert to mix, and incubate at 65°C for 15-30 min, gently inverting to mix every 5 min. Add 350 µL of chloroform and Tris saturated phenol solution to the mixture, vortex to mix, and centrifuge at 12000 r / min for 15 min. Transfer 500 µL of the supernatant to another 2 mL centrifuge tube, add an equal volume of chloroform for extraction again, and centrifuge at 12000 r / min for 15 min. Take 450 µL of the supernatant and transfer it to another 2 mL centrifuge tube. Add an equal volume of chloroform for extraction again, and centrifuge at 12000 r / min for 15 min. Add an equal volume of isopropanol to the supernatant, mix well, and precipitate at -20℃ for 10-15 min; centrifuge at 12000 r / min for 15 min, discard the supernatant and keep the precipitate, wash the precipitate twice with 1 mL of 75% ethanol; dry in an oven at 37℃, dissolve the DNA in 30 µL of deionized water (containing 25 µg / mL RNase A), detect the DNA concentration, and store at -20℃.

[0038] 1.2 Extraction of total RNA from *Plasmodiophora stylosa*

[0039] Cleaned clubroot root material was placed in a pre-chilled mortar and ground into powder under liquid nitrogen freezing conditions. The frozen tissue powder (50-100 mg) was quickly transferred to a 1.5 mL centrifuge tube containing 1 mL of Trizol RNA extraction buffer. The mixture was vigorously vortexed and placed on ice for 5-10 min. Centrifuged at 12000 r / min for 10 min at 4 °C. The supernatant was transferred to another 1.5 mL centrifuge tube, and 200 μL of chloroform was added. The mixture was immediately vigorously vortexed and placed on ice for 5-10 min. After the mixture separated into layers, it was centrifuged at 12000 r / min for 15 min at 4 °C. The supernatant was transferred to another centrifuge tube, and an equal volume of isopropanol was added. The mixture was vortexed and placed on ice for 5-10 min. Centrifuged at 12000 r / min for 10 min at 4 °C. The supernatant was discarded, and 75 μL of isopropanol was added. Add % ethanol, invert to mix and resuspend the precipitate, centrifuge at 4°C and 12000 r / min for 5 min, repeat this step twice; place the centrifuge tube in the centrifuge for 1 min empty, then carefully aspirate the residual liquid with a pipette, and let stand at room temperature for 5-15 min to dry the RNA precipitate; add 30 μL of DEPC water, and after the precipitate dissolves, store at -80°C for later use.

[0040] 1.3 cDNA Synthesis and Target Gene Cloning

[0041] Using Takara Biotech's Prime Scropt 1 st The Strand cDNA synthesis kit was used to synthesize cDNA. The 20 µL mixture was prepared according to the instructions as follows: 2 µg total RNA, 1 µL Oligo(dT)18 Primer (0.5 µg / µL), 10 µL 2X Reaction Mix, 1 µL EasyScript RT / RI Enzyme Mix, 1 µL g DNA Remover, and RNase-free Water, bringing the total volume to 20 µL. The reaction conditions were: incubation at 42°C for 30 min, followed by incubation at 85°C for 5 sec. The synthesized cDNA was stored at -20°C for short-term use in subsequent experiments.

[0042] Primer design: A 300-500 bp CDS sequence (SEQ ID NO.3) was selected as the interference sequence for each gene. The fragments were compared on NCBI (https: / / www.ncbi.nlm.nih.gov / ) to ensure interference specificity. Furthermore, the absence of corresponding interference sites in Arabidopsis was confirmed using the TAIR website (https: / / www.arabidopsis.org / ) to avoid off-target effects (sequences are in the appendix). Homologous arm primers for restriction enzyme sites were automatically generated using the Novizan introductory cloning website (https: / / bio.vazyme.com / gongju.html). The ds1301 vector sequence and the selected gene sequence of approximately 300 bp were input into the website. KpnI and BamHI restriction sites were selected to generate the sense primers, and SacI and SpeI restriction sites were selected to generate the antisense primers (primers are in the appendix).

[0043] PbFAP-102 positive-strand primers:

[0044] p-PbFAP-102-F:5'-cccgtgcagctgcggggtaccACGAACGTATCGTTCCGGG-3'

[0045] p-PbFAP-102-R: 5'-cgcgtacgtaaggttggatccAAGTGCACCATGGTTCACGAT-3'

[0046] PbFAP-102 antisense primers:

[0047] o-PbFAP-102-F: 5'-caattcaattcagtggagctcAAGTGCACCATGGTTCACGAT-3'

[0048] o-PbFAP-102-R: 5'-caggactctagaccccactagtACGAACGTATCGTTCCGGG-3'

[0049] DS1301 vector primers:

[0050] V-1-F: 5'-cgttgagtggccctgtttctc-3'

[0051] V-2-F: 5'-gcttcaaattctaatccccaa-3'

[0052] Gene cloning: Using cDNA / DNA as templates, the target gene was amplified by PCR using gene-specific primers with added homologous arms for cloning. Reaction system: 1 μL Pfu DNA Polymerase, 10 μL Pfu Buffer, 2.5 μL High Pured NTPs, 2 μL cDNA, 1 μL each of primers F and R, and ddH2O to a final volume of 50 μL. Reaction conditions: Pre-denaturation at 95℃ for 2 min, denaturation at 94℃ for 20 s, annealing at 57℃ for 20 s, extension at 72℃ for 15 s / kbp, final extension at 72℃ for 5 min, and storage at 4℃ for 2 min. PCR products were electrophoresed on a 1.2% agarose gel to detect the size of the amplified target gene and to recover bands of uniform size (see TaKaRa's DNA Agarose Gel Electrophoresis Recovery Kit for detailed steps).

[0053] 1.4 Construction of RNAi expression vector for HIGS

[0054] Enzyme digestion products and homologous recombination of the sense strand: The empty vector ds1301 plasmid was double-digested with restriction endonucleases BamHI and KpnI to prepare the following reaction system: 10 µL of ds1301 vector, 5 µL of 10×rCutSmart Buffer, 1 µL of BamHI enzyme, 1 µL of KpnI enzyme, and 33 µL of ddH2O. After preparation, the system was briefly centrifuged and incubated at 37°C for 4 h. Then, the target gene was ligated to the vector via homologous recombination, with the following reaction system: 2 µL of double-digested ds1301 fragment, 1 µL of target gene fragment, 2 µL of Exnase II homologous recombination enzyme, 4 µL of CE II Buffer, and 1 µL of ddH2O. After preparation, the system was briefly centrifuged and incubated at 37°C for 30 min.

[0055] Intermediate vector and antisense strand recombination: The intermediate vector was double-digested with restriction endonucleases SacI and SpeI to prepare the following reaction system: 10 µL of ds1301 vector, 5 µL of 10×rCutSmart Buffer, 1 µL of SacI enzyme, 1 µL of SpeI enzyme, and 33 µL of ddH2O. After preparation, the system was briefly centrifuged and incubated at 37°C for 4 h. Then, the antisense strand was ligated to the intermediate vector via homologous recombination, with the following reaction system: 2 µL of double-digested intermediate vector, 1 µL of antisense strand fragment, 2 µL of Exnase II homologous recombinase, 4 µL of CE II Buffer, and 1 µL of ddH2O. After preparation, the system was briefly centrifuged and the reaction system was placed in a PCR chamber for recombination. The reaction was carried out at 37°C for 30 min before transformation or temporary storage at -20°C. Heat shock transformation of *E. coli* DH5α: 10 μL of ligation product was added to 50 μL of competent *E. coli* cells, incubated on ice for 30 min, heat-shocked at 42 °C for 45 s, incubated on ice for 2 min, then 400 μL of SOC medium was added. After mixing, the cells were incubated at 37 °C at 200 r / min for 1 h. The mixture was then plated on LB agar plates containing the corresponding kanamycin and incubated upside down at 37 °C for 12 h until single colonies appeared. Colony PCR test: A single white colony was picked up with a pipette tip and placed in 10 μL of sterile water. After mixing by pipetting, 1 μL of the mixture was added to 20 μL of Super PCR Mix and mixed thoroughly for PCR. Cycling conditions: (Pre-denaturation: 95 °C, 2 min; Denaturation: 95 °C, 15 s; Annealing: 56 °C, 15 s; Extension: 72 °C, 15 s / kbp; Final extension: 72 °C, 5 min; Storage: 16 °C, 4 min). After single colonies are selected and verified as positive by PCR, they are sent to Qingke Company for sequencing. Those with correct sequencing results are used to extract plasmids for transformation of Agrobacterium.

[0056] 1.5 Agrobacterium-mediated electroporation

[0057] Remove Agrobacterium competent cells from -80℃ and place them on ice for about 5-10 min. Add 1 μL of plasmid to 100 μL of competent cells and mix well. Add the mixture to a clean, dry electroporation cup and let it stand for 1 min. Turn on the electroporator and set it to "Agr". Wipe the surface of the electroporation cup dry and then perform electroporation by pressing "Pulse". Quickly add preheated SOC and place the cup in a shaker at 28℃ and 220 r / min for 30 min to recover and incubate. Remove the cup and spread it on an LB agar plate containing antibiotics. Incubate overnight at 28℃. Identify the cells after colonies have grown.

[0058] 1.6 Agrobacterium-mediated chemical transformation

[0059] Add 5 μL of the final RNAi vector plasmid (approximately 1-2 μg) to 100 μL of Agrobacterium competent cells and mix well; incubate on ice for 10 min, flash freeze in liquid nitrogen for 5 min, then incubate in water at 37℃ for 5 min, followed by an ice bath for 2 min; add 800 μL of liquid LB medium and incubate at 28℃ and 200 rpm for 4-5 h; spread 200 μL of the bacterial culture onto a solid LB culture dish containing 25 mg / L rifampicin and 50 mg / L kanamycin, and incubate at 28℃ for 48 h; after the colonies have grown, pick single clones, and after positive detection confirms successful transformation, the bacterial culture can be used for subsequent gene genetic transformation.

[0060] 1.7 Screening and inoculation experiments of transgenic plants

[0061] Screening of transgenic plants: Rapid DNA extraction method: Take a sample, grind the sample and add 600 uL of rapid extraction buffer, incubate in a 95℃ metal bath for 15 min, then at 10000 r / min for 10 min; after cooling to room temperature, take 10 uL of supernatant, dilute with 60 uL of H2O and it can be used for PCR amplification.

[0062] DNA from transgenic plants was extracted using the rapid DNA extraction method / CTAB method and amplified with specific primers. Plants that produced specific bands were collected as T0 generation seeds. These seeds were then screened in tissue culture flasks containing hygromycin. Plants that grew normally were identified as positive plants, which were then used in disease resistance testing experiments.

[0063] Inoculation with *Plasmodiophora stylosa*: After culturing *Arabidopsis thaliana* or *Brassica napus* for approximately 2 weeks (14 days), use a pipette to take 1 mL of a 1×10⁻⁶ solution. 6 Or 1×10 7 / mL of dormant spores of *Plasmodiophora* were applied to the roots of the plants for inoculation, and the disease index was recorded 21 days after inoculation.

[0064] Clubroot Disease Disease Index Statistics and Grading Standards: The disease index of potted rapeseed was calculated 4 weeks after inoculation with clubroot fungus. The rapeseed was removed from the soil with tweezers, minimizing root damage. After rinsing off the soil, the clubroot disease disease index was calculated. Disease Index (DI) = (1n1 + 3n2 + 5n3 + 7n4) × 100 / 7N t Where n1, n3, and ns represent the number of Arabidopsis plants with disease severity levels of 0, 1, 3, 5, and 7, respectively, and N represents the number of plants in the thalassemia. tThe total number of Arabidopsis thaliana plants is used for disease control. The control effect (%) is calculated as (1 - disease index of transgenic plants / disease index of Col-0) × 100. According to the five-level grading standard, level 0 indicates no disease; level 1 indicates a very small number of tumors mainly on lateral roots, with negligible damage to the main root; level 3 indicates medium to large spherical tumors on the main root; level 5 indicates severe tumors on lateral and main roots, with the main root completely swollen and the fine roots on the lateral roots completely damaged; level 7 indicates completely swollen and rotten roots.

[0065] 1.8 Real-time quantitative PCR (qPCR)

[0066] 1.1 Real-time quantitative PCR was performed using the SYBR Green method. The method was based on the instructions provided with Bio-Rad's iTaq™ Universal SYBR® Green Supermix. Quantitative primers were designed using Beacon Designer 8.0 Real-time PCR primer design software. RNA was extracted from the samples and reverse transcribed into cDNA using 2 µg of RNA. The real-time quantitative PCR (15 µL) reaction system was as follows: Components: iTaq™ Universal SYBR® Green supermix (2X) 7.5 µL, Forward primers (10 µM) 0.5 µL, Reverse primers (10 µM) 0.5 µL, cDNA 0.5 µL, dd H2O 6 µL. Reaction conditions: 95℃, 10 min; 95℃, 15 sec, 57℃, 15 sec, repeated for 45 cycles; 65℃~95℃, increasing by 0.5℃ per cycle for 5 sec, 16℃, 1 min. The reaction was performed on a Bio-Rad CFX 96 instrument. After amplification, 2... -∆∆Ct The method calculates the relative expression level of genes. Primers used in quantitative qPCR experiments:

[0067] The primers for the PbFAP-102 gene are:

[0068] qPCR-PbFAP-102-F: 5'-AGCGTTTGCGTTGAAGGTG-3'

[0069] qPCR-PbFAP-102-R: 5'-CGCGCGATGATCTCCATCT-3'

[0070] The primers for the ACTIN gene of *Plasmodiophora stearothermia* are:

[0071] Pbactin_qF:5'-CACCGACTACCTGATGAA-3'

[0072] Pbactin_qR:5'-CAGCTTCTCCTTGATGTC-3'

[0073] The primers for the rapeseed qBnACTIN7 gene are:

[0074] qBnACTIN7_qF: 5'-CCCTGGAATTGCTGACCGTA-3',

[0075] qBnACTIN7_qR: 5'-TGGAAAGTGCTGAGGGATGC-3'

[0076] 1.9 Plant material, strains and plasmids

[0077] Rapeseed: Zhongshuang 11 (ZS11), commercially available seeds. Plant cultivation conditions: temperature 22℃, humidity 75%. Plant nutrient soil formula: Finnish Kejira peat moss: Jiangsu Peilei substrate: vermiculite = 8:4:1.

[0078] The pathogen, *Cladophora mycoides*, was found in the root system (Williams 1 physiological race) ZJ-1 of clubroot disease in a rapeseed experimental field in Zhijiang, Hubei Province.

[0079] The ds1301 plasmid is disclosed in Host-induced gene silencing offungal-specific genes of Ustilaginoidea virensconfers effective resistance to rice false smut, doi:10.1111 / pbi.13756.

[0080] 2. Experimental Results and Analysis

[0081] 2.1 Construction of PbFAP-102 RNAi vector

[0082] PbFAP-102, which is highly expressed at all stages, was screened from the transcriptome of *Plasmodium styracifolium* ZJ-1. Figure 1 A). Following experimental methods 1.1 and 1.2, RNA was extracted from *Plasmodium*, reverse transcribed into cDNA, and a partial fragment of the flagella-associated protein PbFAP-102 was amplified using the cDNA template. Following experimental methods 1.3 and 1.4, a 300-500 bp target gene fragment was inserted into a vector containing a hairpin structure to construct an RNAi vector. To detect the successful construction of the RNAi vector, positive identification was performed using specific primers for the sense and antisense strands, and the recombinant plasmid was double-digested with KpnI and SpeI for detection. Figure 1 B), successfully constructed one HIGS RNAi vector.

[0083] 2.2 Creation of PbFAP-102 transgenic rapeseed

[0084] The constructed RNAi vector was sent to the company for transformation, resulting in T0 generation transgenic rapeseed seedlings. Normally growing plants were transferred to flowerpots and then transplanted to the transgenic field. Leaves from plants approximately 4 weeks old were collected, DNA was extracted, and positive plants were identified using specific primers and PCR. Figure 2 A) The results showed that 16 PbFAP-102-RNAi transgenic plants were obtained. Seeds from the identified positive transgenic plants were collected and can be used in subsequent transgenic rapeseed disease resistance experiments, such as... Figure 2 As shown in B, these transgenic plants exhibit normal growth phenotypes and do not affect the growth of rapeseed.

[0085] 2.3 PbFAP-102 transgenic plants can improve the resistance of rapeseed to clubroot disease.

[0086] After obtaining transgenic plants, we wanted to verify the resistance of the transgenic material to clubroot disease. Two weeks after the plants began growth, they were inoculated with clubroot fungus, and the root phenotype was observed after five weeks of growth. For example... Figure 3 As shown in Figure A, wild-type rapeseed variety ZS11 (Double 11) exhibits significant root disease, with swollen and even brown taproots, and few or no lateral roots. The disease index is 68.5. Figure 3 A), and the symptoms of root swelling in all HIGS transgenic rapeseed were significantly reduced, with numerous lateral roots. The disease index of PbFAP-102-RNAi-2 and PbFAP-102-RNAi-7 transgenic lines was 48.72 and 46.52, respectively, with control effects of 28.88% and 32.09%. The results indicate that the disease index of HIGS transgenic materials was lower than that of wild type, which can improve the plant's resistance to clubroot. To verify that the improved resistance of transgenic plants to clubroot was due to siRNA silencing of the target gene in clubroot bacteria, we extracted RNA from rapeseed roots 3 weeks after inoculation. qPCR results showed that the expression level of the PbFAP-102 gene in transgenic plants was significantly lower than that of ZS11 ( Figure 3 D), while the root follicle bacteria content of transgenic plants was significantly lower than that of ZS11 ( Figure 3 E), indicating that the transgenic plants have increased resistance to clubroot disease, which is due to the siRNA expressed in the plant inhibiting the expression of the target gene in clubroot bacteria, thereby inhibiting the damage of clubroot bacteria to the plant.

[0087] 3. Summary and Discussion

[0088] Clubroot disease poses a serious threat to the yield and quality of Brassica crops (Brassica oleracea var. spp.) and is a soil-borne disease. Control efforts are intensive, but chemical control is prone to environmental pollution and can easily lead to resistance in clubroot fungi. While biological control is effective, it is costly and difficult to implement in production. Therefore, creating and breeding resistant varieties can provide an effective measure for clubroot control. Flagella in eukaryotes have been extensively studied in protozoa (Trekkissus brucei) and humans, but research on them in clubroot fungi is very limited, and there are currently no reports on them. Flagella, also belonging to eukaryotes, play a significant role in the structure and function of flagella in humans and Trypanosoma brucei. Therefore, we hypothesized that there might be corresponding structures or related modifications in the flagella of clubroot fungi that could affect their reproduction and development. This paper focuses on the function of flagella-related proteins and aims to create related resistant materials. HIGS technology, based on RNAi, has advantages such as good targeting, long-lasting effect, and short cycle, providing a new approach for creating new resistant germplasm. This study constructed a flagellation-associated protein PbFAP-102-RNAi vector and obtained two transgenic plants. The disease resistance test of clubroot showed that they had a good disease resistance phenotype, indicating that flagellation-associated protein PbFAP-102 can be used as a gene resource for green control of clubroot disease in rapeseed.

[0089] References

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Claims

1. PbFAP-102, a flagellation-associated protein from *Plasmodiophora stearothermiae*, characterized in that... The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding flagella-associated protein PbFAP-102 in *Plasmodium* as described in claim 1, characterized in that, Its CDS sequence is shown in SEQ ID NO.

1.

3. The application of a substance that reduces the expression level of flagella-associated protein PbFAP-102 in the clubroot fungus of claim 1 in enhancing plant resistance to clubroot disease.

4. The application according to claim 3, characterized in that, The substance used to reduce the expression level of flagella-associated protein PbFAP-102 in the *Plasmodium* species of claim 1 is selected from host-induced gene silencing RNAi vectors targeting this gene, gene editing systems, siRNA, dsRNA, and ordinary RNAi vectors.

5. The application of negative regulation of the encoding gene described in claim 2 in enhancing plant resistance to clubroot disease.

6. A host-induced gene silencing RNAi vector for silencing the PbATG8 gene as described in claim 2, characterized in that, The host-induced gene silencing RNAi vector uses the CDS sequence fragment shown in SEQ ID NO.3 as the interference sequence.

7. Engineered bacteria containing the RNAi vector of claim 6.

8. Any one of the following applications of the RNAi vector of claim 6 and the engineered bacteria of claim 7: 1) Increase the plant's resistance to clubroot disease; 2) Prepare products that enhance the resistance of plants to clubroot disease; 3) Cultivate plants with improved resistance to clubroot disease; 4) Prepare products that cultivate plants with enhanced resistance to clubroot disease; 5) Improve plants with high resistance to clubroot disease or prepare products made from plants with high resistance to clubroot disease; 7) Inhibits the growth and development of clubroot pathogens; 8) Prepare products that inhibit the growth and development of clubroot bacteria.

9. The application according to claim 8, characterized in that, The plant in question is selected from rapeseed.

10. A method for cultivating plants resistant to clubroot pathogen, characterized in that, The method includes introducing the host-induced gene silencing RNAi vector of claim 6 into a target plant to obtain a clubroot-resistant plant, wherein the clubroot-resistant plant has higher resistance to clubroot than the target plant, and the RNAi vector targets the gene encoding the flagella-associated protein PbFAP-102 in the clubroot fungus of claim 2.

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