Focadh1 protein and application thereof

Abstract

The application discloses a banana fusarium wilt FocADH1 protein and application thereof, and belongs to the field of molecular plant pathology. The application mainly discloses a banana fusarium wilt FocADH1 protein and application thereof as a target in preventing and treating banana fusarium wilt. The application obtains a banana fusarium wilt mutant strain through genetic engineering. The mutant strain is significantly inhibited in growth under osmotic stress, oxidation stress and cell wall stress, and the pathogenicity of the mutant strain to banana seedlings is greatly weakened. Molecular interaction proves that the deubiquitinating enzyme FocUbp8 directly interacts with FocADH1 and regulates the ubiquitination level of FocADH1. Enzyme activity and staining results show that the deletion of FocADH1 leads to the weakening of the antioxidant defense ability of the strain and the abnormal accumulation of active oxygen. The application takes FocADH1 as a target, provides a theoretical basis and an application foundation for screening of anti-banana fusarium wilt drugs, cultivation of disease-resistant varieties and development of green biological pesticides, and has important scientific research and industrial values.

Description

Technical Field

[0001] This invention belongs to the technical field of molecular plant pathology, specifically relating to a FocADH1 protein of the banana wilt pathogen and its application. Background Technology

[0002] Bananas, as a dual-purpose crop (fruit and food), are a major food crop in tropical regions worldwide and also one of my country's important fruits. Banana wilt disease has long been a significant threat to the banana industry, and its pathogen is *Fusarium oxysporum* var. *cubiclesii*. Fusarium oxysporum f. sp. Cuban , Seal The fourth physiological race of banana wilt disease (tropical race 4) emerged in the 1990s. Seal TR4 (a type of cyclophosphamide) has impacted banana cultivation patterns, and as banana seedlings carrying the pathogen circulate globally, the pathogen has spread worldwide, causing severe damage to the banana industry.

[0003] Depending on the variety of bananas infected Seal It is divided into four physiological species, numbered 1-4, with physiological species number 4 further divided into tropical species number 4 (…). Seal tropical race 4, Seal TR4) and subtropical species No. 4 ( Seal subtropicalrace 4, Seal STR4), currently the most pathogenic one. Seal TR4. Because the spores of Fusarium wilt, the causal agent of banana wilt, can survive in the soil for extended periods, and are easily spread through soil, water, seedlings, people, livestock, and machinery, and because the pathogen content in the soil accumulates with continuous cropping, it can easily lead to large-scale outbreaks of Fusarium wilt and hinder banana cropping. Currently, there are no immune banana cultivars or effective control measures, making the control of Fusarium wilt a global research and production challenge.

[0004] Currently, the control of banana wilt disease mainly relies on chemical control, breeding of disease-resistant varieties, and biological control. Chemical control involves using chemical agents to suppress the disease, primarily small-molecule organic antibacterial agents. While using fungicides like carbendazim can control the spread of the disease to some extent in the early stages, the effectiveness drops sharply or even becomes virtually ineffective during the budding stage and subsequent growth stages. Chemical control is not durable, easily lost in the soil, and cannot be effectively preserved there for long periods. Since the wilt pathogen can survive in the soil for 8-10 years, repeated use leads to high control costs and damages the soil environment. Therefore, developing environmentally friendly, efficient, and sustainable biological control technologies has become a pressing research focus. However, chemical control is not durable, easily lost in the soil, and repeated use leads to high costs and soil environmental damage; the breeding cycle for disease-resistant varieties is long, and currently there are no immune cultivars; the effectiveness of biological control is easily affected by environmental factors, resulting in insufficient stability. The root cause of these technical bottlenecks lies in… Seal The infection cycle and pathogenic mechanisms of TR4 are not fully understood, and effective molecular targets are lacking. Although some pathogenic genes have been identified in recent years, research on why these genes affect pathogenicity and the mechanisms of their upstream regulation remains very limited. Therefore, there is an urgent need to explore new targets. Seal The TR4 key pathogenic gene was investigated to elucidate its mechanism of regulating pathogenicity and explore its potential as a target for novel fungicides. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention proposes a FocADH1 protein of banana wilt pathogen and its application.

[0006] The technical solution adopted in this invention: This invention discloses a mutant strain of Fusarium wilt of banana with low pathogenicity, wherein the mutant strain of Fusarium wilt of banana has at least one of the following characteristics: (a) The catalase activity was lower than that of the wild-type strain; (b) The activity of alcohol dehydrogenase was lower than that of the wild-type strain; (c) The growth inhibition rate on culture media containing hydrogen peroxide, sodium chloride, sorbitol, fluorescent whitening agent or Congo red was higher than that of wild-type strain; The mutant strain of Fusarium wilt of banana was obtained by genetic engineering to modify the gene encoding the FocADH1 protein of Fusarium wilt of banana. FocADH1 The coding region CDS sequence was replaced with the hygromycin B resistance gene expression frame, the nucleotide sequence of which is shown in SEQ ID NO.19, and the amino acid sequence of the banana wilt pathogen FocADH1 protein is shown in SEQ ID NO.2. The coding gene of the banana wilt pathogen FocADH1 protein is... FocADH1 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] This invention discloses the application of the mutant strain of Fusarium wilt of banana described above in the preparation of a kit for evaluating the pathogenicity or stress adaptability of Fusarium wilt of banana, wherein the stress is: cell wall stress, cell membrane stress, osmotic stress and oxidative stress.

[0008] The method for constructing the mutant strain of Fusarium wilt of banana includes the following steps: (1) Using the genomic DNA of wild-type Fusarium wilt fungus as a template, amplify the gene encoding the FocADH1 protein of the Fusarium wilt fungus. FocADH1 The CDS sequence and its 5' and 3' flanking homologous sequences; (2) Using the genome of *Fusarium wiltii* and an expression cassette containing the hygromycin B resistance gene as dual templates, the gene encoding the *Fusarium wiltii* FocADH1 protein was expressed by fusion PCR. FocADH1 The coding region CDS sequence was replaced with the hygromycin B resistance gene expression frame to obtain the gene knockout fragment; (3) The gene knockout fragment was converted into a protoplast of Fusarium wilt of banana, and the mutant strain of Fusarium wilt of banana was obtained by resistance screening and PCR identification.

[0009] This invention discloses a method for evaluating the pathogenicity of Fusarium wilt of banana, comprising the following steps: using a mutant strain of Fusarium wilt of banana as a low pathogenicity control strain, using the test strain as an experimental strain, inoculating the conidial suspensions of the control strain and the experimental strain into the root system of banana seedlings respectively, culturing and calculating the disease index, and evaluating the pathogenicity of the strain based on the disease index.

[0010] Preferably, the inoculation method is root wound infection, and the concentration of conidia inoculated is 1×10⁻⁶. 6 spores / mL.

[0011] The disease index is based on the degree of browning of the bulb and is graded as follows: Grade 0 - no browning of the bulb; Grade 1 - brown spots inside the bulb; Grade 2 - browning area inside the bulb is less than 25%; Grade 3 - browning area inside the bulb is 25% to 75%; Grade 4 - browning area inside the bulb is greater than 75%.

[0012] Compared with the prior art, the present invention has the following beneficial effects: Significantly reduced pathogenicity: Bioassay results showed that the pathogenicity of the mutant strain of *Fusarium wilt* was significantly reduced. At the same inoculation concentration (e.g., 1×10⁻⁶), the pathogenicity was significantly reduced. 6Under the same conditions (spores / mL) and treatment, wild-type banana plants exhibited typical leaf yellowing, wilting, and severe browning of the vascular bundles of the rootstock compared to the knockout mutant strain of Fusarium wilt, while the knockout mutant strain only showed mild lesions, indicating that... FocADH1 Genes are essential key genes that maintain the pathogenicity of banana wilt pathogen.

[0013] Significantly reduced adaptability to stress: Compared with the wild type, the mutant strain of *Fusarium wilt* showed no significant difference in growth rate and colony morphology on normal culture media (basal metabolism was unaffected), but its growth was significantly inhibited under various stress conditions. On culture media containing NaCl (osmotic stress), SDS (cell membrane stress), CFW, or CR (cell wall stress), the growth inhibition rate of the mutant strain was significantly higher than that of the wild type, indicating that the deletion of this gene significantly reduced the strain's tolerance to environmental stress and significantly weakened its ability to survive stress.

[0014] Clear genetic background and phenotype: This mutant strain was constructed using precise methods and has a single genetic background. Homologous recombination successfully replaced the CDS sequence in the FocADH1 coding region with the HygR resistance gene. The knockout strain exhibits a highly consistent and stable phenotype, characterized by normal basic growth, sensitivity to environmental stress only, and loss of pathogenicity. This phenotype is not easily affected by culture conditions or passage number, making it an ideal strain for studying the pathogenic mechanism and environmental adaptation molecular mechanisms of Fusarium wilt in bananas.

[0015] Theoretical value and application prospects are broad: This invention provides FocADH1 The gene knockout mutant strain and related validation methods provide a clear gene target for elucidating the pathogenic molecular mechanism and stress response pathway of Fusarium wilt of banana. This gene can serve as a target for screening novel drugs against Fusarium wilt of banana, and also provides a theoretical basis for studying disease-resistant banana varieties through homologous recombination technology or developing green biopesticides targeting this gene, which has important scientific research and potential application value. Attached Figure Description

[0016] Figure 1 Diagram of mutant knockout strategy; Figure 2 A diagram for verifying the knockout mutant; Figure 3 For Δ Focadh1 The verification diagram is backfilled, where Δ Focadh1 -1 represents a gene knockout mutant, Δ Focadh1 / FocADH1 -1、Δ Focadh1 / FocADH1 -2、Δ Focadh1 / FocADH1 -3 represents the different complemented mutant transformants obtained; Figure 4 For Δ Focadh1 Restore the colony growth diagram, where Δ Focadh1 -1 represents a gene knockout mutant, Δ Focadh1 / FocADH1 -1、Δ Focadh1 / FocADH1 -2、Δ Focadh1 / FocADH1 -3 represents the different complemented mutant transformants obtained; Figure 5 For Δ Focadh1 Reinforced colony growth statistics, where Δ Focadh1 -1 represents a gene knockout mutant, Δ Focadh1 / FocADH1 -1、Δ Focadh1 / FocADH1 -2、Δ Focadh1 / FocADH1 -3 represents the different complemented mutant transformants obtained; Figure 6 For Δ Focadh1 Colony diagram of growth medium, where Δ Focadh1 -1、Δ Focadh1 -2 is FocADH1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 7 For Δ Focadh1 Figure 1 shows the growth of carbon-deficient bacterial colonies on Czapek's medium, where Δ Focadh1 -1、Δ Focadh1 -2 is FocADH1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 8 For Δ Focadh1 Colony growth diagrams under different stress conditions, where Δ Focadh1 -1、Δ Focadh 1-2 are FocADH1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 9 For Δ Focadh1 Statistical graph of colony growth inhibition rate under different stress conditions, where Δ Focadh1- 1. Δ Focadh1 -2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 10 For Δ Focadh1 Browning of bulbs, including Δ Focadh1 -1、Δ Focadh1-2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 11 For Δ Focadh1 Pathogenicity analysis, including Δ Focadh1 -1、Δ Focadh1-2 for Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 12 The leaf yellowing status of ΔFocadh1 is shown, where Δ Focadh1 -1、Δ Focadh1 -2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 13 For Δ Focadh1 Leaf wilting rate, where Δ Focadh1 -1、Δ Focadh1 -2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 14 For Δ Focadh1 Leaf lesions, including Δ Focadh1 -1、Δ Focadh1 -2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 15 For Δ Focadh1 Leaf lesion area statistics, including Δ Focadh1 -1、Δ Focadh1 -2 is Focadh1 Knockout mutants of different transformants, Δ Focadh1 / FocADH1 To replace the mutant; Figure 16 For Focusup 8 self-activation verification; Figure 17 Point-to-point verification between Focubp8 and Focadh1; Figure 18 Deubiquitination of adh1 by ubp8; Figure 19 For Δ Focadh1 Catalase activity assay; Figure 20 For Δ Focadh1 Detection of alcohol dehydrogenase activity; Figure 21 For Δ Focadh1 Malondialdehyde (MDA) detection in banana roots 48 hours after infection; Figure 22 For Δ Focadh1 Malondialdehyde (MDA) detection in banana bulbs 48 hours after infection; Figure 23 For Δ Focadh1 Banana roots were stained with DCFH-DA fluorescence 48 hours after infection; Figure 24 The results are quantitative statistical results of DCFH-DA fluorescent staining of banana roots 48 h after infection. Detailed Implementation

[0017] To make the inventive objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings: In order to better understand the above-mentioned objectives, features, and advantages of this invention, the advantages of this invention will be further illustrated below by comparing the embodiments with the accompanying drawings and specific implementation methods.

[0018] The purpose of this invention is to overcome the shortcomings of insufficient understanding of the key pathogenic genes and control targets of Fusarium wilt in bananas, and to provide a Fusarium wilt pathogen with significantly reduced pathogenicity. FocADH1 Gene knockout mutants, and their application in pathogenicity detection and green control research of banana wilt, provide new gene targets for the biological control and disease-resistant breeding of banana wilt.

[0019] This invention proposes a FocADH1 protein from the banana wilt pathogen and its application. The specific implementation method is as follows:

[0020] Example 1 FocADH1 Gene knockout and replacement 1.1 Gene knockout Protoplasts of Fusarium wilt of banana (Foc) were prepared according to standard experimental protocols, and fungal transformation was performed. A gene replacement vector for obtaining the FocADH1 gene knockout mutant was constructed using two-fragment amplification PCR technology. Two pairs of primers, ADH1-LB-CK / LB-R and ADH1-RB-F / RB-CK, were used to amplify the 5' and 3' flanking sequences of the FocADH1 CDS sequence using Phanta Super polymerase (Vazyme, P501-1). Then... Seal TR4 and HPT are dual templates, amplified using two pairs of primers: ADH1-LB-F / HPT-R1 and ADH1-RB-R / HPT-F1. FocADH1The DNA fragment was replaced by the hygromycin B resistance gene (HygR). The purified gene knockout fragment (3-5 µg) or plasmid (10 µg) was added to a centrifuge tube containing 1 mL of protoplasts, inverted to mix, and incubated on ice in the dark for 30 min. 1 / 3 volume of PTC (approximately 330 µL) was added dropwise to the centrifuge tube, gently inverted to mix, and incubated at room temperature in the dark for 20 min. The transformed mixture was then transferred to a centrifuge tube containing 10-15 mL of RM medium and incubated at 80-110 rpm, 25-28 °C for 15 h. The revived mycelial mixture was added to a CM solid culture medium containing hygromycin at a suitable temperature (lower layer antibiotic concentration 100 μg / mL, upper layer antibiotic concentration 300 μg / mL), and cultured at 28℃ for 2-3 days. Transformants were picked for further verification, and the knockout mutant was confirmed by PCR using primers ADH1-LB-CK / Hpt-up, ADH1-RB-CK / Hpt-down, and ADH1-InF / InR. The results are as follows. Figure 1 , Figure 2 As shown.

[0021] Table 1. List of primers used for gene knockout

[0022] 1.2 Gene replacement When performing gene complementation experiments, the gene is obtained through PCR amplification. FocADH1 The gene coding sequence and its natural promoter sequence were used to ligate the target fragment into the pKNT vector using a one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd., China). The recombinant complement vector was first verified for fragment correctness using PCR before being sent for sequencing. After sequencing verification, it was introduced into the ΔpKNT vector. FocADH1 Positive transformants were obtained from the knockout mutant strains through PCR screening combined with growth phenotype assays, and the results are as follows: Figure 3 , Figure 4 , Figure 5 As shown.

[0023] Table 2 List of gene complementation primers

[0024] Example 2: Fusarium wilt pathogen of banana FocADH1 Phenotypic analysis and pathogenicity determination of genes 2.1 Culture medium formulation For evaluation Seal TR4 (wild-type, WT), two mutant strains (Δ) FocADH1 -1 and Δ FocADH1-2) To investigate phenotypic differences among complements, strains were inoculated into PDA, CM, and Cz media and cultured at 28°C for 5 days, and colony growth rates were observed. To study the differences in stress response among strains, 0.7 M sodium chloride (NaCl), 1.0 M sorbitol, 0.1 mg / ml fluorescent whitening agent (CFW), 0.2 mg / ml Congo red, 0.005% sodium dodecyl sulfate (SDS), and 10 mM hydrogen peroxide (H2O2) were added to CM solid medium, and the pH of the medium was adjusted to 5.8, 6.8, and 7.8, respectively. After culturing at 28°C for 3-5 days, the vertical colony diameter was measured. The inhibition rate (IR) was finally calculated using the following formula, where "D" is the diameter of the control sample and "d" is the diameter of the treated sample: IR = (D − d) / D × 100%.

[0025] 2.2 Analysis of Growth Culture Medium Results On various growth media, Δ FocADH1 The mutant strain had a slightly smaller colony diameter than the wild type (WT), while the replacement strain reverted to a phenotype consistent with WT. However, cross-sections on nitrogen-deficient medium showed that WT had significantly more aerial hyphae than the Δ strain. FocADH1 Mutant strains indicate that this gene may play a role in energy metabolism, as shown in the results. Figure 6 , Figure 7 As shown.

[0026] 2.3 Analysis of results from stress culture medium Stress susceptibility assay results showed that, compared with wild-type (WT) and complement strains, Δ FocADH1 The knockout mutant showed significantly increased growth inhibition rates under cell wall stress (CFW and CR), cell membrane stress (SDS), osmotic stress (sorbitol and NaCl), and oxidative stress (H2O2). Notably, the mutant was particularly sensitive to CFW, NaCl, and H2O2, with inhibition rates exceeding 20%, while the wild type and complement showed very low growth inhibition. Under pH (5.8, 6.8, 7.8) stress, the mutant also exhibited mild growth disadvantage. These results indicate that FocADH1 is a key factor in maintaining the tolerance of *Fusarium wilt* f. sp. banana to multiple abiotic stresses, especially cell wall integrity and oxidative and osmotic stresses. Figure 8 , Figure 9 As shown.

[0027] 2.4 Pathogenicity determination 2.4.1 Inoculation method for damaged roots Take a bacterial strain that has grown on a plate for about 5 days, and chop it into 1 cm pieces. 2After collecting the mycelial blocks near the edge, incubate with PDB for 3-5 days. Filter to obtain spores, then adjust the concentration to 10. 6 1 spore / ml; 5-6 leaf stage Brazilian banana seedlings (AAA) were removed from small pots, rinsed to remove soil, and the roots were trimmed. Approximately 5 cm of the roots were cut off with scissors, and the seedlings were immediately immersed in the above spore solution. Soaking time was 1 hour, with the solution shaken every 10 minutes. Ten seedlings were inoculated with each strain. After inoculation, the banana seedlings were transplanted back to the same small pots and cultured in a greenhouse at 28 ± 2℃, 16 hours light, 8 hours dark, and 60%–85% relative humidity, with normal watering or fertilization. Leaf yellowing was observed and photographed after 10, 15, and 20 days. After 30 days, the seedlings were pulled out, the bulbs were longitudinally cut, and the disease severity was recorded. Each experiment was repeated three times.

[0028] The severity of the disease is graded as follows: 0 (no browning on the bulb), 1 (some brown spots on the inner bulb), 2 (browning on less than 25% of the inner bulb area), 3 (browning on up to 3 / 4 of the inner bulb), and 4 (the entire inner bulb is dark brown and the plant is dead). Calculate the disease index. Disease index = 100 × ∑ (number of bulbs at each level × severity at each level) / (total number of bulbs surveyed × highest severity level).

[0029] 2.4.2 Leaf inoculation method Leaf inoculation: Leaves were surface-sterilized with 75% ethanol and rinsed with sterile water. Mycelial blocks with a diameter of 5 mm were inoculated onto the leaves, and lesions were observed after 3 days of incubation at 28°C. All experiments were performed in triplicate. For banana leaf inoculation experiments, fresh mycelial blocks (5 mm in diameter) of each strain were inoculated onto banana leaves surface-sterilized with 75% ethanol. Disease progression was assessed 5 days after inoculation. All experiments were performed in triplicate.

[0030] 2.4.3 Analysis of pathogenicity results Banana seedlings inoculated with wild-type (WT) and reinjected strains showed severe leaf yellowing and wilting after 30 days, with large areas of dark brown necrosis visible in longitudinal sections of the corms (disease index mostly concentrated between 2 and 4). Meanwhile, two independent knockout mutants (Δ...) Focadh1 -1、Δ Focadh1 -2) The inoculated plants grew healthily, with bulbs remaining mostly white or showing only slight browning (disease index concentrated at level 0-1), showing no significant difference from the H2O control (CK) group. The disease index distribution of the reinjected strain returned to the wild-type level, as shown in the results. Figure 10 , Figure 11 , Figure 12 , Figure 13 As shown.

[0031] 2.4.4 Analysis of Leaf Inoculation Results In banana leaf inoculation trials, the wild-type (WT) banana produced necrotic lesions covering an area of ​​0.91 cm². 2 The lesion area of ​​the reinjected strain recovered to 0.84 cm². 2 The two knockout mutants produced significantly smaller lesion areas on the leaves, only 0.54 cm². 2 about.

[0032] Conclusion: The combined results of the above root and leaf inoculation indicate that... FocADH1 Gene knockout severely weakened the pathogenicity of Fusarium wilt of banana, while reintroduction restored pathogenicity, proving that this gene is a key factor essential for the infection and pathogenicity of Fusarium wilt of banana. Figure 14 , Figure 15 As shown.

[0033] Example 3 3.1 Mutual Verification The FocUBP8 sequence (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.18) was inserted into the pGBKT7 vector to construct the BD-FocUBP8 bait plasmid, which was then transformed into competent Saccharomyces cerevisiae Y2HGold cells as bait strains for yeast two-hybrid (Y2H) screening. To identify the interaction target of FocUBP8, the constructed BD-UBP8 bait plasmid and pGADT7 vector were first transformed into yeast strain Y2HGold, and the presence of self-activation was observed on SD / -Trp / -Leu and SD / -Trp / -Leu / -His / -Ade plates. The vector sequence is as follows: Table 3 Vector Sequence

[0034] The specific steps are as follows: 1) Set up three experimental groups: the first group is pGBKT7-53 and pGADT7-T, the second group is pGBKT7-Lam and pGADT7-T, and the third group is BD and pGADT7. The plasmid concentration in each group is at least 100 ng. 2) Add 5 μL of pre-denatured carrier DNA and 300 μL of 1×PEG / LiAc solution to each reaction tube and mix well; 3) Add 50 μL of resuspended yeast competent cells to each reaction tube and mix slowly; 4) Incubate in a 30℃ water bath for 30 minutes, mixing every 10 minutes; 5) Add 20 μL of DMSO to each tube and mix well; 6) Heat shock in a 42℃ water bath for 15 minutes, inverting the container every 5 minutes to mix thoroughly; 7) Centrifuge at 700 g for 5 min; 8) Discard the supernatant and resuspend the medium in 1 ml of YPD Plus liquid medium; Note: YPD Plus is a specially formulated enhanced transformation medium; YPD medium is not used in this step.

[0035] 9) Incubate at 30℃ in a shaker for an appropriate time (1 h); 10) Centrifuge at high speed for 15 seconds; 11) Discard the supernatant, resuspend the cells in 0.9% NaCl solution, and observe whether self-activation occurs. The results are as follows: Figure 16 As shown, no self-activation phenomenon was confirmed.

[0036] After confirming the absence of self-activation, yeast two-hybrid screening was performed. A cDNA library containing *Fusarium wilt*, the causal agent of banana wilt, was constructed in *Saccharomyces cerevisiae* Y187 cells and co-transformed into the Y187 strain. The co-transformation procedure followed the self-activation transformation procedure. After co-transformation, the bacterial culture was plated on SD / -Trp / -Leu / -His / -Ade plates for initial screening. Yeast colonies from the initial screening plates were then transferred to a secondary screening medium, SD / -Trp / -Leu / -His / -Ade / X-α-Gal, for secondary screening. The selected colonies were validated by PCR and sent to Shanghai Baisai Biotechnology Co., Ltd. for sequencing. Analysis of the results led to the preliminary screening of candidate proteins. The candidate protein gene sequences were inserted into pGADT7 for point-to-point validation. The vectors used are shown in the table below. Table 4 Vector Sequence

[0037] like Figure 17 As shown, all three can grow normally on SD-Leu-Trp medium, indicating that the plasmid transformation was successful. However, negative strains cannot grow normally on SD-Leu-Trp-His-Ade medium, but positive strains and BD-Focubp8 / AD-Focadh1 can grow normally with the addition of X-α-Gal and produce blue colonies, indicating that the two interact.

[0038] 3.2 Ubp8 Deubiquitination of ADH1 To further determine whether ADH1 undergoes deubiquitination in banana wilt, we transformed the FLAG-ADH1 recombinant vector into wild-type and Δubp8 mutants, respectively. The transformants were inoculated into PDB medium and cultured at 28°C and 180 rpm for 2 days, then fungal mycelial samples were collected. Approximately 0.2 g of the ground mycelium was added to 200 μL of protease lysis buffer, placed on ice, and 1% PMSF and 1% protease inhibitor were added. Each sample was vortexed 2-3 times. After vortexing, the samples were placed on ice, with approximately 5 minutes between each vortex. The samples were then centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatant was collected.

[0039] 1) Magnetic bead pretreatment Gently pipette the Anti-Flag immunomagnetic beads to fully suspend them, and place 25–50 μL of the bead suspension into a 1.5 mL centrifuge tube.

[0040] Add 500 μL of IP Lysis / Wash Buffer and mix gently with a pipette. Gently pipette to resuspend the Anti-Flag immunomagnetic beads and place them on a magnetic separator. Once the solution becomes clear, discard the supernatant with a pipette. Repeat the washing process twice.

[0041] 2) Sample binding Add the protein sample containing the Flag tag to the pretreated magnetic beads and incubate on a flip mixer (at room temperature for 1-2 hours, or at 4°C overnight). Place the above mixture on a magnetic rack and let it stand for about 1 minute until the solution becomes clear. Then transfer the supernatant to a new centrifuge tube for later use (the supernatant can be used to detect whether there is any residue of the Flag tag protein). The remaining contents in the original centrifuge tube are the protein-magnetic bead complex. Note: During the bonding process, the magnetic beads may clump together or appear in sheets, which is normal and will not affect the experimental results.

[0042] 3) Washing Add 1000 μL of IP Lysis / Wash Buffer to the protein-magnetic bead complex obtained in the above steps, gently mix the magnetic beads for 5-10 min, then let it stand on a magnetic rack for about 1 min until the solution becomes clear, and then aspirate the supernatant. Repeat the above steps twice until the OD280 of the supernatant after washing is less than 0.05; Note: If the OD280 of the supernatant is still greater than 0.05, increase the number of washes as appropriate.

[0043] 4) Elute with SDS-PAGE loading buffer This experiment requires direct detection of the target protein. Therefore, 80–100 μL of 1xSDS-PAGE loading buffer was added to the washed protein-magnetic bead complex, boiled for 10 min, cooled to room temperature, and the magnetic beads were separated on a magnetic rack. The supernatant was then used for SDS-PAGE analysis. After enrichment and purification with anti-FLAG magnetic beads, Western blot analysis was performed using an anti-ubiquitin antibody to detect ubiquitinated FLAG-ADH1. The results showed that, compared with the wild type, the ubiquitination level of ADH1 was Δ ubp8 Significantly elevated in mutants, results as follows Figure 18 As shown.

[0044] 3.3 Enzyme activity assay 3.3.1 Detection of catalase (CAT) activity This experiment used a catalase (CAT) activity assay kit (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC0205). CAT (EC 1.11.1.6) is widely found in animals, plants, microorganisms and cultured cells. It is the most important H2O2 scavenging enzyme and plays an important role in reactive oxygen species scavenging systems.

[0045] H2O2 has a characteristic absorption peak at 240 nm. CAT can decompose H2O2, causing the absorbance of the reaction solution at 240 nm to decrease with reaction time. The activity of CAT can be calculated based on the rate of change of absorbance.

[0046] Required instruments and supplies: UV spectrophotometer / ELISA reader, benchtop centrifuge, adjustable pipette, micro-quartz cuvettes / 96-well UV plates, mortar / homogenizer, ice and distilled water.

[0047] Operating steps: I. Sample Processing Tissue preparation: Weigh approximately 0.1 g of tissue according to tissue mass (g), add 1 mL of extraction buffer, and homogenize in an ice bath. Centrifuge at 8000g at 4℃ for 10 min, collect the supernatant, and place on ice for analysis.

[0048] II. Measurement Procedure 1. Preheat the microplate reader for at least 30 minutes, adjust the wavelength to 240 nm, and zero the instrument with distilled water.

[0049] 2. Before the test, bathe the CAT test working solution in a 25°C water bath for at least 10 minutes.

[0050] 3. Add 10 μL of sample and 190 μL of working solution to a 96-well UV plate, mix immediately and start timing. Record the initial absorbance value A1 at 240 nm for 5 s and the absorbance value A2 after 1 min 5 s. Calculate ΔA = A1 - A2.

[0051] III. CAT Activity Calculation 3. Calculated based on sample quality: Definition of unit: One unit of enzyme activity is defined as the ability of one gram of tissue to catalyze the degradation of 1 μmol of H2O2 per minute in a reaction system.

[0052]

[0053] Vreactiontotal: Total volume of the reaction system, 2 × 10-4 L; ε: Molar extinction coefficient of H2O2, 43.6 L / mol / cm; d: Optical path length of the 96-well UV plate, 0.6 cm 3.3.2 Results Analysis The enzyme activity assay results showed that Δ Focadh1 The catalase (CAT) activity of the -1 mutant was significantly decreased compared to the wild-type (WT) (P=0.0034, a decrease of approximately 38%), confirming that... FocADH1 Genes are key regulatory factors in maintaining the normal antioxidant defense capacity and hydrogen peroxide scavenging function of strains, as shown in the results. Figure 19 As shown.

[0054] 3.3.3 Detection of alcohol dehydrogenase activity 3.3.3.1 Experimental Reagents and Methods Alcohol dehydrogenase (ADH) kit (catalog number: G0874W, microplate method, 96 samples); Alcohol dehydrogenase (ADH, EC 1.1.1.1) is present in many organisms. In humans and many other animals, it breaks down toxic alcohols. In yeast and many bacteria, some alcohol dehydrogenases catalyze the reverse reaction as part of fermentation. This kit utilizes alcohol dehydrogenase to catalyze the reaction of acetaldehyde and NADH to produce ethanol and NAD+. The activity of alcohol dehydrogenase is calculated by detecting the rate of decrease of NADH at 340 nm.

[0055] Required instruments and supplies: microplate reader, 96-well plate, benchtop centrifuge, adjustable pipette, mortar and pestle, ice and distilled water.

[0056] 3.3.3.2 Assay of alcohol dehydrogenase (ADH) activity Sample preparation: Tissue sample: Weigh approximately 0.1 g of tissue, add 1 mL of extraction buffer, and homogenize in an ice bath. Centrifuge at 12000 rpm, 4℃ for 10 min, collect the supernatant, and place on ice for testing.

[0057] [Note]: If the sample size is increased, extraction can be performed at a ratio of tissue mass (g): extraction liquid volume (mL) of 1:5~10.

[0058] Detection procedure: ① Preheat the microplate reader for at least 30 minutes and adjust the wavelength to 340 nm. ② Place the reagents in a 37℃ water bath for 5 minutes. ③ Add the reagents to the 96-well plate according to the table below. For the test tubes: Sample 40, Reagent 1 10, Reagent 2 130, Reagent 3 20. Mix well and immediately read the A1 value at 340 nm. After 5 minutes at room temperature (25℃), read the A2 value. ΔA = A1 - A2.

[0059] [Note]: (1) If ΔA is too small, the sample volume V1 can be increased (e.g., increased to 60 μL, then reagent 2 should be reduced accordingly), or the reaction time T can be extended (e.g., 10 min or longer). The adjusted sample volume V1 and reaction time T need to be substituted into the calculation formula and recalculated. (2) If the value of ΔA is greater than 0.2, the sample volume V1 needs to be reduced (e.g., reduced to 20 μL, then reagent 2 should be increased accordingly), or the reaction time T needs to be shortened (e.g., 2 min or shorter). The adjusted sample volume V1 and reaction time T need to be substituted into the calculation formula and recalculated.

[0060] Results Calculation: Calculated based on sample protein concentration: Enzyme activity definition: One enzyme activity unit is defined as the amount of enzyme that consumes 1 nmol NADH per minute per milligram of tissue protein. ADH (nmol / min / mg prot) = [ΔA ÷ (εxd) x 10] 9 xV2]÷(V1×Cpr)÷T=321.54×ΔA÷Cpr V1---Sample volume added, 0.04 mL; d---Optical path length of 96-well plate, 0.5 cm; V2---Total volume of reaction system, 2 × 10⁻⁶ -4 L;ε --- NADH molar extinction coefficient, 6.22 × 10⁻⁶ 3 L / mol / cm; T---reaction time, 5 min; Cpr---protein concentration, mg / mL.

[0061] 3.3.4 Results Analysis The results of the alcohol dehydrogenase (ADH) activity assay showed that Δ Focadh1 The -1 mutant showed a highly significant decrease in ADH activity compared to the wild-type (WT) (P=0.0003), a reduction of over 50%. This biochemical phenotype directly confirms the gene's... FocADH1 The absence of [certain substances] severely impairs the reductive metabolic pathway of acetaldehyde to ethanol, resulting in [the following]. Figure 20 As shown.

[0062] 3.3.5 Malondialdehyde (MDA) Detection The measurement of MDA is often combined with the measurement of SOD. The level of SOD activity indirectly reflects the body's ability to scavenge oxygen free radicals, while the level of MDA indirectly reflects the severity of free radical attack on the body's cells. The analysis of SOD and MDA results is helpful for the development of medicine, biology, pharmacology and industrial and agricultural production.

[0063] Malondialdehyde (MDA) test kit (purchased from Nanjing Jiancheng Bioengineering Institute, item number: A003-1) 1. Required instruments and reagents: Visible spectrophotometer or ELISA reader, constant temperature water bath or boiling water kettle adjustable to about 95℃, centrifuge, vortex mixer, 10 mL centrifuge tubes, distilled water, anhydrous ethanol (small amount), glacial acetic acid (analytical grade, concentration ≥99.5%), 50% acetic acid aqueous solution (can be obtained by diluting glacial acetic acid with distilled water at a ratio of 1:1), chloroform, distilled water, physiological saline, etc.

[0064] 2. Plant tissue samples: Accurately weigh the plant tissue at a ratio of weight (g):volume (mL) = 1:4, add 4 times the volume of homogenizing medium (recommended 0.01 mol / L pH 7.0~7.4 phosphate buffer), mechanically homogenize under ice-water bath conditions to prepare a 20% homogenate, centrifuge at 3500~4000 rpm for 10 minutes, and collect the supernatant for analysis.

[0065] 3. On-machine testing Table 5. Reagents and Contents for Instrumental Testing

[0066] Cap the centrifuge tubes and poke a small hole in the cap with a needle (if you are sure the cap will not pop open during the boiling water bath, you can skip this step). Vortex to mix, incubate at 95°C for 40 minutes, remove and cool with cold water, then centrifuge at 3500-4000 rpm for 10 minutes. Collect the supernatant (if the supernatant is not clear enough, add 0.1 mL of chloroform, vortex for about 60 seconds, and then centrifuge). Measure the OD value of each tube at 532 nm with a 1 cm optical path length, zero the plate with distilled water, and use a spectrophotometer (or add 200 μL of supernatant from each tube to a 96-well plate and read the value at 532 nm using a microplate reader).

[0067] Note: ① Generally, only 1 to 2 standard tubes and blank tubes need to be tested per batch. If the sample does not have special color, turbidity or other phenomena, the control tube can be omitted and the blank tube can be used to replace the control tube for calculation.

[0068] ② When aspirating the supernatant for colorimetric analysis, do not aspirate the precipitate at the bottom. If solid matter remains in the supernatant after centrifugation, increase the centrifugation speed or add 0.1 mL of chloroform, vortex for 60 seconds, centrifuge again, and then aspirate the supernatant.

[0069] ③ When reading the microplate reader, simply add 200 μL of supernatant to the 96-well plate and read the value at 532 nm.

[0070] 4. Calculation formula MDA content (nmol / g tissue) = [(Measured OD - Control OD) ÷ (Standard OD - Blank OD) × Standard concentration (10 nmol / mL) × Total volume of homogenate (mL)] ÷ Tissue weight (g).

[0071] 3.3.6 Results Analysis The malondialdehyde (MDA) test results showed that Δ Focadh1 The abnormal accumulation of MDA in the roots and bulbs of the -1 mutant was significantly higher than that in the wild type (P<0.0001). This result corroborates the previous finding of decreased CAT activity, jointly demonstrating that... FocADH1 Gene deletion severely weakens the strain's ability to scavenge reactive oxygen species (ROS), causing irreversible oxidative damage and membrane system disruption during infection, resulting in... Figure 21 , 22 As shown.

[0072] 3.4 Reactive Oxygen Species (ROS) Staining In plant-pathogen interactions, one of the important defenses of host plants against pathogen invasion is the "oxygen burst," which involves the production of large amounts of reactive oxygen species (ROS) to kill invading pathogens. Wild-type and knockout strains were stained using a reactive oxygen species detection kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: S0033S). Under bright field (BF) conditions, the wild-type (WT) and Δ... Focadh1 The mutant hyphae showed no significant differences in morphology and septal structure; however, under fluorescence, the wild-type hyphae did not show a significant green fluorescence signal, while Δ Focadh1 The mutant hyphae exhibit a high-intensity green fluorescence distribution. This result visually demonstrates that... FocADH1 Gene deletion led to a massive and abnormal accumulation of reactive oxygen species (ROS) within the cell. Combined with previous studies (significantly reduced CAT activity and significantly increased MDA content in the mutant), this microphenotype further confirms the intracellular redox homeostasis imbalance induced by gene knockout, as shown in the following results. Figure 23 , 24 As shown.

[0073] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

Claims

1. A mutant strain of Fusarium wilt of banana with low pathogenicity, characterized in that, The mutant strain of banana wilt pathogen has at least one of the following characteristics: (a) The catalase activity was lower than that of the wild-type strain; (b) The activity of alcohol dehydrogenase was lower than that of the wild-type strain; (c) The growth inhibition rate on culture media containing hydrogen peroxide, sodium chloride, sorbitol, fluorescent whitening agent or Congo red was higher than that of wild-type strain; The mutant strain of Fusarium wilt of banana was obtained by genetic engineering to modify the gene encoding the FocADH1 protein of Fusarium wilt of banana. FocADH1 The coding region CDS sequence was replaced with the hygromycin B resistance gene expression frame, the nucleotide sequence of which is shown in SEQ ID NO.19, and the amino acid sequence of the banana wilt pathogen FocADH1 protein is shown in SEQ ID NO.

2. The coding gene of the banana wilt pathogen FocADH1 protein is... FocADH1 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application of the mutant strain of Fusarium wilt of banana according to claim 1 in the preparation of a kit for evaluating the pathogenicity or stress adaptability of Fusarium wilt of banana, wherein the stress is: cell wall stress, cell membrane stress, osmotic stress and oxidative stress.

3. The method for constructing a mutant strain of *Fusarium wilt* fungus according to claim 1, characterized in that, Includes the following steps: (1) Using the genomic DNA of wild-type Fusarium wilt fungus as a template, amplify the gene encoding the FocADH1 protein of the Fusarium wilt fungus. FocADH1 The CDS sequence and its 5' and 3' flanking homologous sequences; (2) Using the genome of *Fusarium wiltii* and an expression cassette containing the hygromycin B resistance gene as dual templates, the gene encoding the *Fusarium wiltii* FocADH1 protein was expressed by fusion PCR. FocADH1 The coding region CDS sequence was replaced with the hygromycin B resistance gene expression frame to obtain the gene knockout fragment; (3) The gene knockout fragment was converted into a protoplast of Fusarium wilt of banana, and the mutant strain of Fusarium wilt of banana was obtained by resistance screening and PCR identification; The amino acid sequence of the FocADH1 protein of the banana wilt pathogen is shown in SEQ ID NO.2, and the gene encoding the FocADH1 protein of the banana wilt pathogen is also shown. FocADH1 The nucleotide sequence is shown in SEQ ID NO.

1.

4. A method for evaluating the pathogenicity of Fusarium wilt of bananas, characterized in that, The method includes the following steps: using the mutant strain of banana wilt pathogen as described in claim 1 as a low pathogenicity control strain, and using the test strain as an experimental strain, inoculating the conidial suspensions of the control strain and the experimental strain into the root system of banana seedlings, respectively, and calculating the disease index after cultivation, and evaluating the pathogenicity of the strain based on the disease index.

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