Application of protein FocVPS41 in regulating the pathogenicity of Fusarium wilt in bananas

Abstract

This invention discloses the application of the protein FocVPS41 in regulating the pathogenicity of Fusarium wilt of banana. The invention utilizes homologous recombination to knock out the FocVPS41 gene from Fusarium wilt of banana, obtaining the knockout mutant ΔFocVPS41. Experimental results show that, compared to Foc4, ΔFocVPS41 has a smaller colony diameter and significantly reduced sporulation, decreased tolerance to the cell wall stress factor Congo red, and increased tolerance to the osmotic pressure regulator sorbitol and the pesticide cyazofamid. The absence of ΔFocVPS41 significantly reduces the pathogenicity of Foc4. This invention confirms that FocVPS41 is essential for the production of conidia in Fusarium wilt of banana, its response to cell wall stress, osmotic pressure stress, the fungicide cyazofamid, and its pathogenicity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of protein FocVPS41 in regulating the pathogenicity of Fusarium wilt of banana. Background Technology

[0002] Banana wilt, also known as Panama disease or yellow leaf disease, is a soil-borne fungal epidemic caused by *Fusarium oxysporum* f.sp. cubense (Foc). It is highly prevalent and difficult to control, especially race 4 (Foc TR4), which has caused devastating damage to the banana industry. Thoroughly exploring the pathogenic genes of *Fusarium wilt* and studying their functions will help to comprehensively understand the molecular mechanisms of its pathogenesis and provide a theoretical basis for the integrated control of banana wilt.

[0003] VPS41 is an important component of the HOPS complex (Homotypic fusion and vacuole sorting). FocVPS41 is highly conserved in Fusarium, but its specific function in Fusarium wilt of banana remains unclear. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to disclose a new function of the banana wilt fungus gene FocVPS41 and the protein FocVPS41. The coding region of the gene FocVPS41 consists of the nucleotide sequence shown in positions 206 to 652, 702 to 728, 778 to 1769, 1822 to 3565, and 3620 to 4426 of SEQ ID NO: 1, and the protein FocVPS41 encoded by it is the protein shown in SEQ ID NO: 2. This invention utilizes a split-marker strategy to amplify upstream and downstream homologous fragments of the hygromycin resistance gene in FocVPS41 via PCR, and introduces these fragments into protoplasts of *Fusarium oxysporum*. The FocVPS41 gene is then knocked out of *Fusarium oxysporum* using homologous recombination, resulting in the knockout mutant ΔFocVPS41. A gene complementation vector is constructed and introduced into the ΔFocVPS41 protoplasts. The gene is then randomly inserted back into the knockout mutant, resulting in the complemented mutant ΔFocVPS41-Com. This knockout mutant exhibits defects in conidia production, reduced colony diameter, decreased tolerance to the cell wall stress factor Congo red, and increased tolerance to the osmolarity regulator sorbitol and the pesticide cyazofamid. Pathogenicity assays show that the knockout mutant ΔFocVPS41 has significantly reduced pathogenicity, while the pathogenicity of the complemented mutant ΔFocVPS41-Com is restored to the Foc4 level. The above experiments demonstrate that FocVPS41 of Fusarium wilt of banana is a pathogenic gene associated with Fusarium wilt of banana.

[0005] The technical solution of this invention mainly includes the following:

[0006] This invention provides the application of protein FocVPS41 in regulating the pathogenicity of Fusarium wilt of banana, the amino acid sequence of which is shown in SEQ ID NO:2; the application is to reduce the pathogenicity of Fusarium wilt of banana by knocking out the gene encoding protein FocVPS41 or inhibiting the expression of the gene encoding protein FocVPS41.

[0007] The expression of the gene encoding the knockout protein FocVPS41 or the gene encoding the repressor protein FocVPS41 can be achieved by using substances targeting the gene or by genetic engineering techniques.

[0008] Furthermore, the gene encoding the protein FocVPS41 consists of the nucleotide sequence shown in SEQ ID NO: 1, positions 206 to 652, 702 to 728, 778 to 1769, 1822 to 3565, and 3620 to 4426.

[0009] Furthermore, the application is the use of the protein FocVPS41 in inhibiting the growth and development of Fusarium wilt in bananas.

[0010] Furthermore, the application is the use of the protein FocVPS41 in reducing the sporulation rate of Fusarium wilt in bananas.

[0011] Furthermore, the application is the use of the protein FocVPS41 in reducing the resistance of Fusarium wilt in bananas to cell wall stress.

[0012] Furthermore, the cell wall stress is Congo red stress.

[0013] This invention provides the application of protein FocVPS41 as a target for a drug used to control plant diseases, specifically banana wilt caused by Fusarium wilt.

[0014] Furthermore, the aforementioned Fusarium wilt species is Fusarium wilt species race 4 (Foc4).

[0015] The present invention also relates to the application of protein FocVPS41 in regulating the resistance of Fusarium wilt of banana to osmotic stress and to cyazofamid, wherein the application is to improve the resistance of Fusarium wilt of banana to osmotic stress by knocking out gene FocVPS41 or inhibiting gene expression of FocVPS41.

[0016] The present invention also relates to a FocVPS41 gene knockout mutant of Fusarium wilt of banana, which is obtained by knocking out the gene encoding the protein FocVPS41.

[0017] Compared with the prior art, the significant effects achieved by the present invention are as follows:

[0018] This invention provides a novel function for the protein FocVPS41 and its encoding gene. The coding region of the gene FocVPS41 consists of the nucleotide sequence shown in SEQ ID NO: 1, positions 206 to 652, 702 to 728, 778 to 1769, 1822 to 3565, and 3620 to 4426. The protein it encodes, FocVPS41, is the protein shown in SEQ ID NO: 2. The FocVPS41 protein contains three conserved domains: “SCOP d1fwxa2”, “Clathrin”, and “RING”. This invention replaces the FocVPS41 gene with the hygromycin phosphotransferase (HPH) gene, obtaining the Foc4 knockout mutant ΔFocVPS41. Experiments showed that compared to Foc4, ΔFocVPS41 has a smaller colony diameter and significantly reduced sporulation, decreased tolerance to the cell wall stress factor Congo red, and increased tolerance to the osmotic pressure regulator sorbitol and the pesticide cyazofamid. Pathogenicity tests showed that the deletion of FocVPS41 significantly reduced the pathogenicity of Foc4; after the gene was reintroduced, its pathogenicity was restored. This invention confirms that FocVPS41 is essential for the production of conidia in *Fusarium wiltii*, its response to cell wall stress, osmotic pressure stress, the fungicide cyazofamid, and its pathogenicity. Our research contributes to elucidating the pathogenic molecular mechanism of *Fusarium wiltii* and provides a target gene for the development of effective fungicides. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the knockout and complementation vector construction of the FocVPS41 gene in Fusarium wilt of banana; in the diagram, A: schematic diagram of FocVPS41 gene knockout construction, B: schematic diagram of FocVPS41 gene complementation vector construction.

[0020] Figure 2 This is an agarose gel electrophoresis image of the PCR amplification products of outside and inside primers for some candidate FocVPS41 gene knockout transformants.

[0021] Figure 3 This is an agarose gel electrophoresis image of the PCR amplification products of HYG primers, NEO primers, and FocVPS41inside primers for some candidate FocVPS41 gene complement transformants.

[0022] Figure 4 This is a Southern hybridization analysis of the FocVPS41 gene knockout and complement transformants from Fusarium wilt of banana; in the figure, A: gel electrophoresis before Southern hybridization and membrane transfer, B: Southern hybridization of the FocVPS41 gene knockout and complement transformants.

[0023] Figure 5This image shows the colony morphology of FocVPS41 gene knockout and complement transformants from Fusarium wilt of banana. In the image, A: Colony morphology of FocVPS41 gene knockout and complement transformants after 5 days of culture on PDA; B: Hydrophobicity of FocVPS41 gene knockout and complement transformants colonies; C: Longitudinal section of FocVPS41 gene knockout and complement transformant colonies; D: Growth curve of FocVPS41 gene knockout and complement transformants.

[0024] Figure 6-A , Figure 6-B , Figure 6-C Microscopic observation of FocVPS41 gene knockout transformants; Figure 6-A : FocVPS41 gene knockout transformant hyphal tips, conidial clusters, and conidia; Figure 6-B Sporulation yield determination of FocVPS41 gene knockout transformants; Figure 6-C Determination of conidial size in FocVPS41 gene knockout transformants.

[0025] Figure 7-A , Figure 7-B This is an analysis of FocVPS41 gene knockout transformants under different stress conditions; Figure 7-A Colony diameter; Figure 7-B Colony growth inhibition rate.

[0026] Figure 8-A , Figure 8-B , Figure 8-C This is a pathogenicity analysis of FocVPS41 gene knockout and complement transformants; Figure 8-A Symptoms of disease in Brazilian banana seedlings one month after inoculation with FocVPS41 gene knockout and complement transformants; Figure 8-B Disease index; Figure 8-C Determination of relative fungal growth in diseased bulbs of Brazilian bananas after inoculation. Detailed Implementation

[0027] To better understand the technical content of this invention, the invention will be further described below with reference to specific embodiments.

[0028] 1. Experimental Materials

[0029] 1.1 Test strains and plants

[0030] The tested strain was physiological race 4 of Fusarium wilt of banana (Foc4), and the tested plant was a banana (Cavendish, AAA) with 5-6 leaves (plant height 10-15cm).

[0031] 1.2 Host bacteria and plasmid vectors

[0032] The host bacteria were *Escherichia coli* DH5α strain and yeast XK-125 (a generous gift from Professor Zhang Haifeng of Nanjing Agricultural University). The vector was pKOV21 (a generous gift from Professor Chen Xiaolin of Huazhong Agricultural University), and the gene complementation vector was pYF11-NEO (a generous gift from Professor Zhang Chengqi of Anhui Agricultural University).

[0033] 2. Experimental Methods

[0034] 2.1 Amplification of upstream and downstream homologous fragments of the *Fusarium oxysporum* FocVPS41 gene

[0035] The FocVPS41 gene knockout in Fusarium wilt of banana was performed using a split-marker strategy to amplify upstream and downstream homologous fragments, such as... Figure 1 As shown, primers were designed by selecting approximately 3kb sequences upstream and downstream of the FocVPS41 gene (Table 1).

[0036] Table 1. Primers for FocVPS41 gene knockout and complementation

[0037]

[0038]

[0039] The split-marker strategy was used to amplify the upstream and downstream homologous recombination fragments of FocVPS41 by PCR. Two rounds of PCR amplification were performed. In the first round of PCR amplification: using Foc4 genomic DNA as a template, PCR amplification was performed with primers VPS41-LBCK and VPS41-LB-R to obtain the homologous arm VPS41-LB1 of the FocVPS41 gene; PCR amplification was performed with primers VPS41-RB-F and VPS41-RBCK to obtain the homologous arm VPS41-RB1 of the FocVPS41 gene; and PCR amplification was performed using pKOV21 plasmid DNA as a template with primers HYG-F and HYG-R to obtain the hygromycin resistance gene fragment HYG-1.4kb. The products of the first round of PCR amplification were analyzed using OMEGA Gel Extraction. The DNA was purified and recovered using a kit, and the concentration was determined using a Nanodrop-2000c. The recovered product was diluted to 50 ng / μL and used as template DNA for the second round of PCR amplification. The second round of PCR amplified homologous recombination fragments containing the HYG partial sequence of the FocVPS41 gene. Specifically, using VPS41-LB1 and HYG-1.4kb as templates, and primers VPS41-LB-F and HYG-R1, Over-Lap PCR amplification was performed to obtain the upstream homologous recombination fragment VPS41-HYG-LB2 of the FocVPS41 gene; using VPS41-RB1 and HYG-1.4kb as templates, and primers HYG-F1 and VPS41-RB-R, Over-Lap PCR amplification was performed to obtain the downstream homologous recombination fragment VPS41-HYG-RB2 of the FocVPS41 gene.

[0040] The PCR reaction system for the first round was as follows:

[0041] Components volume <![CDATA[ddH2O]]> up to 50.0μL 2xTaq Master Mix (Novazime P111) 25.0μL Foc4 DNA (100 ng / μL) 1.0μL VPS41-LBCK / VPS41-RBCK(10μmol / L) 2.0μL VPS41-LB-R / VPS41-RB-F(10μmol / L) 2.0μL

[0042] The first round of PCR reaction conditions were: 95℃ for 5 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 2 min, for a total of 35 cycles; 72℃ for 5 min. The PCR amplification products were purified and recovered using the OMEGA Gel Extraction Kit.

[0043] The second round of PCR reaction system is as follows:

[0044] Components volume <![CDATA[ddH2O]]> up to 50.0μL 2x Phanta Max Buffer (Novazia, P505) 25.0μL dNTP Mix (10mM each) 1.0μL VPS41-LB-F / HYG-F1 (10 μmol / L) 2.0μL HYG-R1 / VPS41-RB-R (10 μmol / L) 2.0μL VPS41-LB1 / VPS41-RB1 (50ng / μL) 1.0μL HYG-1.4kb (50ng / μL) 1.0μL Phanta Max Super-Fidelity DNA Polymerase 1.0μL

[0045] The second round of Over-Lap PCR reaction conditions were: 95℃ for 3 min; 95℃ for 15 sec, 58℃ for 45 sec, 72℃ for 2 min, for a total of 32 cycles; and 72℃ for 5 min. The PCR amplification products were purified and recovered using the OMEGA Gel Extraction Kit.

[0046] 2.2 Amplification of the complement fragment of the FocVPS41 gene from Fusarium wilt of banana

[0047] Construction of the complementation vector for the FocVPS41 gene of Fusarium wilt of banana as follows Figure 1 As shown in Table 1, the promoter sequence 1.8 kb upstream of the FocVPS41 gene and the sequence downstream of FocVPS41 with the stop codon removed were selected to design primers VPS41-Native-1F and VPS41-6045R.

[0048] Using Foc4 genomic DNA as a template, PCR amplification was performed with primers VPS41-Native-1F and VPS41-6045R to obtain the FocVPS41 gene complement fragment FocVPS41-Com-6.0kb; the fragment was purified and recovered using the OMEGA Gel Extraction Kit, and the DNA concentration was determined using Nanodrop-2000c.

[0049] The PCR reaction system is as follows:

[0050] Components volume <![CDATA[ddH2O]]> up to 50.0μL 2x Phanta Max Buffer (Novazia, P505) 25.0μL dNTP Mix (10mM each) 1.0μL VPS41-Native-1F (10 μmol / L) 2.0μL VPS41-6045R (10 μmol / L) 2.0μL Foc4 DNA (100 ng / μL) 1.0μL Phanta Max Super-Fidelity DNA Polymerase 1.0μL

[0051] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 15 sec, 60℃ for 15 sec, 72℃ for 4 min, for a total of 32 cycles; 72℃ for 5 min. The PCR amplification products were purified and recovered using the OMEGA Gel Extraction Kit.

[0052] 2.3 Construction of the complementation vector for the FocVPS41 gene of Fusarium wilt of banana

[0053] pYF11-NEO plasmid DNA was linearized by single-digestion with XhoI. The linearized pYF11-NEO(XhoI) plasmid DNA was recovered by gel excision. The pYF11-NEO(XhoI) plasmid DNA and the complement fragment FocVPS41-Com-6.0kb were co-transformed into yeast XK-125 competent cells at a molar ratio of 1:9. The preparation and transformation of yeast XK-125 competent cells were performed according to the Alkali-cationyeast transformation kit (MP Biomedicals, catalog). The yeast single clones obtained by transformation were verified by PCR using primers pYF11-seq-R and VPS41-CKF (Table 1). The verification complement vector pYF11-NEO-FocVPS41 was used to extract yeast plasmids using a yeast plasmid extraction kit. The yeast plasmids were transformed into Escherichia coli DH5α for propagation. pYF11-NEO-FocVPS41 plasmid DNA was extracted again and stored at -20℃ for later use.

[0054] 2.4 Preparation of Foc4 protoplasts

[0055] Foc4 was inoculated into PDB medium and cultured at 28℃ and 150 rpm for 3–5 days. 5 mL of Foc4 was filtered through three layers of sterile lens paper into fresh PDB medium and cultured at 28℃ and 150 rpm for 12–16 hours. The culture was then filtered through three layers of sterile lens paper and washed 3–5 times with 0.7 mol / L NaCl solution (osmotic stabilizer) to obtain fresh mycelium. An appropriate amount of 10 g / L protoplastase was added at a ratio of enzyme solution to mycelium (volume to mass ratio 10:1), and the mixture was hydrolyzed at 30℃ and 100 rpm for 3–4 hours to obtain protoplast hydrolysate. The hydrolysate was centrifuged at 4000 × g for 10 min at 4℃, and the supernatant was discarded. The precipitate was resuspended in 10 mL of pre-cooled STC solution (containing 10 mmol / L Tris-HCl (pH 7.5), 1.2 mol / L sorbitol, and 50 mmol / L CaCl2); the precipitate was centrifuged, and the supernatant was discarded. Add 1–3 mL of pre-cooled STC solution to resuspend the precipitate, obtaining a Foc4 protoplast suspension, bringing the final protoplast concentration to approximately 2 × 10⁻⁶. 7 Protoplasts per mL were collected and aliquoted into 2.0 mL EP tubes on ice and stored at -80°C for later use.

[0056] The protoplasts of the banana wilt bacterium FocVPS41 knockout mutant were prepared following the same procedure as the preparation of the Foc4 protoplasts described above.

[0057] 2.5 PEG-mediated Foc4 protoplast transformation

[0058] After thawing 200 μL of Foc4 protoplasts on ice, add 3-5 μg of upstream homologous recombination fragments VPS41-HYG-LB2 and VPS41-HYG-RB2 respectively, gently mix, and incubate on ice for 30-60 min; add 1 mL of SPTC (40% PEG 4000, 10 mmol / L Tris HCl, pH 7.5, 1.2 mol / L sorbitol, 50 mmol / L CaCl2) dropwise, mix well, and incubate on ice for 20-30 min; add 25 mL of pre-chilled SPTC, mix well; centrifuge at 4000 rpm for 15 min at 4 °C; discard the supernatant, leaving 5 mL of the mixture, add 5 mL of RM liquid regeneration medium (205.5 g / L sucrose, 3 g / L yeast extract, 20 g / L glucose) to resuspend the precipitate, and incubate at 28 °C with shaking at 100 rpm for 12-16 h. Centrifuge at 4000 rpm for 15 min at 4°C. Carefully and gently remove 5 mL of supernatant. Add 25 mL of RM solid regeneration medium (205.5 g / L sucrose, 3 g / L yeast extract, 20 g / L glucose, 100 μg / mL hygromycin or 200 μg / mL neomycin G418), and gently mix (avoid generating excessive air bubbles). Pour into 3-5 petri dishes and place in a clean bench for 30 min until completely solidified. Then, pour in another 10 mL of medium. RM solid regeneration medium (containing 100 μg / mL hygromycin) was incubated upside down at 28°C for 5–10 days. Single colonies grown on the regeneration medium were transferred to PDA medium (containing 200.0 g potato, 20.0 g anhydrous glucose, 15.0 g agar, and distilled water to a final volume of 1 L) containing 50 μg / mL hygromycin (or 200 μg / mL neomycin G418) and incubated at 28°C for 3–5 days. Single colony hyphae were collected for PCR verification and identification.

[0059] Obtaining FocVPS41 complement transformants: Mix pYF11-NEO-FocVPS41 plasmid DNA (15-20 μg) with 200 μL of ΔFocVPS41 knockout mutant protoplasts. Then follow the same transformation process as the FocVPS41 gene knockout described above. Select transformants for resistance to 200 μg / mL neomycin G418.

[0060] 2.6 PCR validation analysis of FocVPS41 gene knockout mutant

[0061] Genomic DNA was extracted from the wild-type strain Foc4 and the candidate FocVPS41 gene knockout mutant using CTAB, and PCR verification analysis was performed. PCR amplification of the FocVPS41 gene outside fragment was performed using primers VPS41-Outside-1F / VPS41-Outside-4762R; PCR amplification of the FocVPS41 gene inside fragment was performed using primers VPS41-inside-F / VPS41-inside-1138R.

[0062] The PCR reaction system is as follows:

[0063] Components volume <![CDATA[ddH2O]]> up to 20.0μL 2xTaq Master Mix (Novazime P111) 10.0μL Foc4 DNA / ΔFocVPS41 DNA (100ng / μL) 1.0μL VPS41-Outside-1F / VPS41-inside-F(10μmol / L) 1.0μL VPS41-Outside-4762R / VPS41-inside-1138R(10μmol / L) 1.0μL

[0064] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 5 min. The obtained PCR amplification products were analyzed by agarose gel electrophoresis.

[0065] 2.7 PCR validation analysis of the FocVPS41 gene complementation mutant

[0066] Genomic DNA was extracted from the wild-type strain Foc4 and the candidate FocVPS41 gene knockout mutant using CTAB, and PCR verification analysis was performed. PCR amplification of the HYG gene was performed using primers HYG-F / HYG-R; PCR amplification of the NEO gene was performed using primers NEO-probe-F / NEO-probe-563R; and PCR amplification of the inside fragment of the FocVPS41 gene was performed using primers VPS41-inside-F / VPS41-inside-1138R.

[0067] The PCR reaction system is as follows:

[0068]

[0069]

[0070] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 1 min, for a total of 35 cycles; 72℃ for 5 min. The obtained PCR amplification products were analyzed by agarose gel electrophoresis.

[0071] 2.8 Southern hybridization analysis of FocVPS41 gene knockout and complement mutants

[0072] Genomic DNA was extracted from the Foc4, ΔFocVPS41 gene knockout mutant, and ΔFocVPS41-Com gene complement mutant of Fusarium wilt of banana using the CTAB method. 20 μg of genomic DNA was linearized by single-cell digestion with HindIII restriction endonuclease and Southern hybridization was performed according to the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche). The target gene FocVPS41 probe was amplified using primers VPS41-inside-F / VPS41-inside-1138R, the HYG probe was amplified using primers HYG-Probe-1F / HYG-Probe-598R, and the NEO probe was amplified using primers NEO-Probe-F / NEO-Probe-563R (primers are shown in Table 1).

[0073] The PCR amplification system for the DNA probe is as follows:

[0074] Components volume <![CDATA[ddH2O]]> up to 50.0μL 2xTaq Master Mix (Novazime P111) 25.0μL Foc4 DNA(100ng / μL) / pKOV21(10ng / μL)DNA / pKOV21 DNA(10ng / μL) 1.0μL VPS41-inside-F / HYG-Probe-F / NEO-probe-F(10μmol / L) 2.0μL VPS41-inside-1138R / HYG-Probe-598R / NEO-probe-563R(10μmol / L) 2.0μL

[0075] The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 5 min. The obtained PCR amplification products were subjected to agarose gel electrophoresis, and the gel was excised and recovered using the OMEGA agarose gel extraction kit and stored at -20℃ for later use.

[0076] 2.9 Observation of colony morphology and microstructure of FocVPS41 gene knockout and complement mutants

[0077] (1) Colony morphology observation and growth rate determination. Foc4, ΔFocVPS41, and ΔFocVPS41-Com were inoculated onto PDA medium and incubated upside down at 28℃. The colony diameter was measured using the cross-crossing method at 1, 3, and 5 days, and the colony morphology was observed. 20 μL of ddH2O was added to the colonies to analyze their hydrophobicity. Simultaneously, the colonies were longitudinally cut with a scalpel to observe the density of aerial hyphae. Each treatment was replicated in triplicate.

[0078] (2) Microscopic observation of hyphae, and determination of conidia yield and size. *Fusarium oxysporum* f. banana was inoculated into PDA and PDB media, respectively, and cultured at 28°C. The PDA strain was incubated statically for 5 days, while the PDB strain was cultured at 150 rpm. After 5 days, the microstructure of the hyphal tips and conidiophores was observed under a microscope. The length and width of the conidia were measured using imaging software provided with the microscope, and the size of the conidia was determined. The number of conidia was counted using a hemocytometer. Each treatment was performed in triplicate.

[0079] 2.10 Analysis of the abiotic stress response of FocVPS41 gene knockout and complement mutants

[0080] Foc4, ΔFocVPS41, and ΔFocVPS41-Com were inoculated into MM medium containing different stress factors (containing 1.2 mol / L sorbitol, 5 mmol / L sorbitol, etc., respectively). The bacteria were cultured in H2O2, 0.02% (w / w) SDS, 100 μg / mL fluorescent whitening agent (CFW), 100 μg / mL Congo red (CR), and 20 μg / mL cypermethrin (Phenamacril) at 28°C for 5 days. MM medium (2.28 g / L dipotassium hydrogen phosphate, 1.36 g / L potassium dihydrogen phosphate, 0.53 g / L ammonium sulfate, 0.15 g / L sodium chloride, 0.49 g / L magnesium sulfate heptahydrate, 0.07 g / L calcium chloride dihydrate, 0.0025 g / L ferric sulfate heptahydrate, 1.98 g / L glucose, 15 g / L agar powder) as a blank control. The colony diameter was measured using the cross-cross method, and the colony growth inhibition rate under different stress conditions was calculated. Each treatment was replicated in triplicate.

[0081] 2.11 Pathogenicity analysis of FocVPS41 gene knockout and complement mutants

[0082] Take five-leaf stage Brazilian bananas and use conidia (2 × 10⁻⁶) of Foc4, ΔFocVPS41, and ΔFocVPS41-Com respectively. 6 After soaking the damaged roots in a suspension of (number of bacteria / mL) for 60 min, the seedlings were transplanted into nutrient soil. Each strain was inoculated into 30 Brazilian seedlings. The seedlings were cultured in a plant culture room at 28±1℃. After 30 days, the disease incidence on the leaves and bulbs was observed. The bulbs were cut open to observe brown necrotic lesions, and the disease severity and disease index were calculated. Genomic DNA was extracted from the bulbs. Using the genomic DNA from diseased bulbs as a template, primers FocTR4-242-F / FocTR4-242-R were used to specifically detect the amount of Fusarium wilt bacteria in the bulbs, and primers MusaActin-F / MusaActin-R were used as a reference gene (primers are shown in Table 1). The relative biomass of Fusarium wilt bacteria in the diseased tissues was detected by quantitative real-time qRT-PCR.

[0083] 3 Results and Analysis

[0084] 3.1 Obtaining the homologous fragment of the FocVPS41 gene knockout in Fusarium wilt of banana

[0085] Using a split-marker strategy and Foc4 genomic DNA as a template, the first round of PCR amplification yielded a 2114 bp fragment upstream of the FocVPS41 gene (VPS41-LB1) and a 2103 bp fragment downstream of the FocVPS41 gene (VPS41-RB1). The second round of Overlap PCR amplification yielded a 2627 bp fragment upstream of the FocVPS41 gene (VPS41-HYG-LB2) and a 2255 bp fragment downstream of the FocVPS41 gene (VPS41-HYG-RB2).

[0086] 3.2 Screening for the knockout mutant ΔFocVPS41

[0087] Using homologous recombination, the upstream homologous arm VPS41-HYG-LB2 and the downstream homologous arm VPS41-HYG-RB2 of the FocVPS41 gene were transformed into Foc4 protoplasts of *Fusarium wiltii*, yielding 135 hygromycin-resistant transformants. DNA was extracted, and PCR validation analysis was performed on the 135 hygromycin-positive transformants using outside and inside primers of the FocVPS41 gene. The results showed that, due to the substitution of the FocVPS41 gene by the HYG gene in *Fusarium wiltii*, the amplified fragment size using outside primers was 4762 bp in the wild-type Foc4, while the amplified fragment size of the candidate positive knockout mutant was 1815 bp. Simultaneously, inside primer amplification only amplified a specific fragment of 1138 bp in the wild-type Foc4. The validation results for transformants 101, 105, and 123 are as follows: Figure 2 As shown.

[0088] 3.3 Screening of the complementation mutant ΔFocVPS41-Com

[0089] The complement vector pYF11-NEO-FocVPS41 was transformed into protoplasts of the ΔFocVPS41 knockout mutant via PEG-mediated transformation. Seven neomycin G418 resistant transformants were obtained through neomycin G418 resistance selection. Using the genomic DNA of these seven candidate transformants as templates, PCR verification was performed using three primer pairs: HYG-F / HYG-R, NEO-probe-F / NEO-probe-563R, and VPS41-inside-F / VPS41-inside-1138R. The results showed that three NEO-resistant transformants (numbered 01, 02, and 04) were ΔFocVPS41-Com positive complement transformants. Figure 3 ).

[0090] 3.4 Southern hybridization verification of gene knockout mutant ΔFocVPS41 and complement mutant ΔFocVPS41-Com

[0091] Southern hybridization analysis was performed on the candidate knockout mutants ΔFocVPS41 (101, 105, 123) and the complement mutant ΔFocVPS41-Com (01, 02, 04), which had been preliminarily validated by PCR. The results showed that when using the target gene FocVPS41 as a probe, no hybridization bands were observed in any of the three knockout transformants, while hybridization bands were observed in the wild-type Foc4 and all three complement transformants. When using hph as a probe, single-copy bands appeared in both the three knockout and three complement transformants. When using the neomycin gene NEO as a hybridization probe, a hybridization band signal was only observed upstream of the three complement transformants. These experiments further confirmed that all three knockout and three complement transformants were positive transformants. Figure 4 ).

[0092] 3.5 Determination of colony morphology and diameter of gene knockout mutant ΔFocVPS41 and complement mutant

[0093] Foc4, the knockout mutant ΔFocVPS41 (ΔFocVPS41 123), and the complement mutant ΔFocVPS41-Com (ΔFocVPS41-123-Com-04) were inoculated into PDA medium. Colony morphology and diameter were observed 5 days after inoculation. The results showed that compared to Foc4, the colonies of ΔFocVPS41 were significantly smaller than those of Foc4, while the complement mutant recovered to the level of wild-type Foc4. Figure 5 -A, Figure 5 -D), ΔFocVPS41 colonies exhibit reduced hydrophobicity, resulting in a more hygroscopic state; however, the hydrophobicity of the replacement mutant was not fully restored. Figure 5 -B), longitudinal section observation of aerial hyphae showed that, compared to the abundant aerial hyphae of Foc4, the knockout mutant ΔFocVPS41 had fewer aerial hyphae, and the hyphae adhered closely to the surface of the PDA medium. In contrast, the replenishment mutant colonies showed a return to Foc4-like aerial hyphae. Figure 5 -C).

[0094] 3.6 Microscopic observation and sporulation analysis of gene knockout mutant ΔFocVPS41 and complement mutant

[0095] Foc4, the knockout mutant ΔFocVPS41 (ΔFocVPS41 123), and the complement mutant ΔFocVPS41-Com (ΔFocVPS41-123-Com-04) were inoculated into PDA and PDB media, respectively, and sporulation was analyzed after 5 days of culture. The results showed that the mycelia at the edge of ΔFocVPS41 123 colonies were dense and twisted, while the number of conidiophores and corresponding conidiophores per unit area in the central part was significantly reduced. Statistical analysis of the corresponding yields showed that, regardless of whether the culture conditions were static PDA or shaking PDB, the sporulation of ΔFocVPS41 123 was significantly less than that of Foc4 (approximately 100-fold difference). Figure 6-A , Figure 6-B Conidia size analysis showed that the average size of Foc4 conidia was 5.89±3.30μm×2.22±0.50μm, and the average size of ΔFocVPS41123 conidia was 9.07±4.27μm×2.48±0.45μm, which were not significantly different from those of wild-type Foc4. Figure 6-C ).

[0096] 3.7 Response analysis of gene knockout mutant ΔFocVPS41 and complement mutant to different abiotic stress conditions

[0097] Foc4, the knockout mutant ΔFocVPS41 (ΔFocVPS41 123), and the complement mutant ΔFocVPS41-Com (ΔFocVPS41-123-Com-04) were inoculated into MM medium containing different stress factors. The results showed that, compared with Foc4, ΔFocVPS41 was more sensitive to Congo red, but not sensitive to fluorescent whitening agents, sorbitol, and the pesticide cyazofamid. There was no significant difference in sensitivity to SDS and H2O2. Figure 7-A (7-B) indicates that FocVPS41 plays an important role in Foc4's response to cell wall stress, osmotic pressure stress, and tolerance to the pesticide cyazofamid.

[0098] 3.8 Pathogenicity analysis of gene knockout mutant ΔFocVPS41 and complement mutant

[0099] Using the root-damage inoculation method, conidial solutions of Foc4, ΔFocVPS41 (ΔFocVPS41-123), and ΔFocVPS41-Com (ΔFoc VPS41 123Com 04) were inoculated into Brazilian bananas, and observations were conducted after 30 days. The results showed that, compared to the negative control H2O treatment, Brazilian banana seedlings inoculated with Foc4 and ΔFocVPS41-Com conidial solutions were stunted overall, with some plants dying, and the lower leaves showing yellowing, with the yellowing area covering approximately 45–65% of the leaf area. Longitudinal sections of diseased Brazilian banana seedlings revealed dark brown lesions, with the browned area covering approximately 35–70% of the seedling area. However, 30 days after inoculation with ΔFocVPS41 spore solution, there was no significant difference in plant height between the Brazilian banana plants treated with ΔFocVPS41 and those treated with H2O. The lower leaves showed yellowing covering approximately 15–30% of the leaf area. Longitudinal sections of diseased Brazilian banana seedlings also showed browning covering approximately 15–30% of the seedling area. Figure 8-A The disease severity was statistically analyzed, and the calculated ΔFocVPS41 disease index was 24.3, significantly lower than the 84.8 of the wild-type strain Foc4. Figure 8-B Meanwhile, the relative fungal biomass of the corresponding Fusarium wilt fungus was detected in the diseased bulbs. The results showed that the relative fungal biomass of ΔFocVPS41 was significantly smaller than that of Foc4( Figure 8-C This indicates that knocking out the FocVPS41 gene significantly reduces the pathogenicity of Fusarium wilt in bananas.

[0100] In summary, the FocVPS41 gene and its encoded protein provided by this invention can be used for the prevention and control of plant diseases, particularly banana wilt caused by *Fusarium wilt*. Furthermore, the FocVPS41 gene and its encoded protein provided by this invention can serve as targets for drugs used to control plant diseases such as banana wilt. Those skilled in the art can follow the teachings and inspirations of this specification to develop drugs for the prevention and control of plant diseases, especially banana wilt.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a protein FocVPS41 in regulating pathogenicity of Fusarium oxysporum f. sp. cubense, wherein the amino acid sequence of the protein FocVPS41 is shown as SEQ ID NO: 2; and the use is to reduce the pathogenicity of Fusarium oxysporum f. sp. cubense by knocking out or inhibiting the expression of a gene encoding the protein FocVPS41.

2. Use according to claim 1, characterized in that, The gene encoding the protein FocVPS41 consists of the nucleotide sequences shown as positions 206-652, 702-728, 778-1769, 1822-3565, and 3620-4426 in SEQ ID NO:

1.

3. Use according to claim 1, characterized in that, The use is the use of the protein FocVPS41 in inhibiting the growth and development of Fusarium oxysporum f. sp. cubense.

4. Use according to claim 1, characterized in that, The use is the use of the protein FocVPS41 in reducing the sporulation of Fusarium oxysporum f. sp. cubense.

5. The use according to claim 1, characterized in that, The use is the use of the protein FocVPS41 in reducing the resistance of Fusarium oxysporum f. sp. cubense to cell wall stress, wherein the cell wall stress is Congo red stress.

6. Use according to claim 1, characterized in that, The Fusarium oxysporum f. sp. cubense is Fusarium oxysporum f. sp. cubense Race 4.

7. Use of the protein FocVPS41 in the regulation of the resistance to osmotic stress in Fusarium oxysporum f. sp. cubense, characterized in that, The amino acid sequence of the protein FocVPS41 is shown as SEQ ID NO: 2, and the use is to improve the resistance of Fusarium oxysporum f. sp. cubense to osmotic stress by knocking out or inhibiting the expression of a gene encoding the protein FocVPS41.

8. Use of the protein FocVPS41 in the regulation of the resistance of banana fusarium wilt to cyanofenpham. characterized in that, The amino acid sequence of the protein FocVPS41 is shown as SEQ ID NO: 2, and the use is to improve the tolerance of Fusarium oxysporum f. sp. cubense to cayenne by knocking out or inhibiting the expression of a gene encoding the protein FocVPS41.

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