Fusarium oxysporum f. sp. cubense secreted protein FoXYG1 and application thereof

Abstract

The application discloses a banana fusarium wilt secreted protein FoXYG1 and application thereof, and belongs to the technical field of molecular plant pathology. The secreted protein FoXYG1 can induce plant defense response, including causing plant cell hypersensitive necrosis, inducing callose accumulation and up-regulated expression of a plant immune-related pathway marker gene, activating SA, JA, ET and HR pathway-mediated defense pathways to induce plant immune response, and can significantly improve the resistance of plants to fusarium oxysporum and / or fusarium oxysporum. A new way is provided for improving plant disease resistance and reducing disease occurrence, and thus has a wide application prospect in the green development of modern agriculture.

Description

Technical Field

[0001] This invention belongs to the field of molecular plant pathology technology, specifically relating to the secretory protein FoXYG1 of the banana wilt pathogen and its application. Background Technology

[0002] Banana wilt, also known as banana Panama disease, is a soil-borne fungal disease that causes vascular bundle necrosis. The pathogen is *Fusarium oxysporum* f.sp. *cubense* (Foc), a typical soil-borne plant pathogen that invades the host through wounds in the roots and spreads through the vascular bundles to the pseudostem and upper parts of the leaves, blocking the xylem blood vessels and ultimately causing the host to wilt and die, seriously threatening the development of the banana industry.

[0003] Biological stress caused by pathogens is a major factor leading to plant diseases. In the interaction between these two factors, the plant immune response triggered by pathogens manifests at two levels. When pathogens infect plants, their pathogen-associated molecular patterns (PAMPs) induce a basal immune response, also known as the primary immune response (PTI). To obtain the nutrients needed for growth and reproduction from the plant, pathogens secrete a large number of effectors to suppress the basal immune response (PTI), thus suscepting the plant and promoting pathogen infection and disease development—this is known as the immune response stimuli (ETS). However, some pathogen-secreted effectors can be recognized by receptors present in the plant, inducing a higher-level immune response, also known as an ETI.

[0004] The cell wall is a natural barrier for plants to defend against pathogens. During the interaction between pathogens and plants, pathogens secrete large amounts of cell wall degrading enzymes (CWDEs) to depolymerize the polysaccharide components of the host plant's cell wall, disrupting the cell wall structure and allowing pathogens to successfully infect the host by overcoming the epidermal barrier. In addition to acting as virulence factors, CWDEs also function as PAMPs, triggering an immune response in plants. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a secretory protein FoXYG1 from Fusarium wilt of bananas; another objective of this invention is to provide the encoding gene of the secretory protein FoXYG1 from Fusarium wilt of bananas; and yet another objective of this invention is to provide applications of the secretory protein FoXYG1 from Fusarium wilt of bananas.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A secreted protein FoXYG1 of the banana wilt pathogen has the amino acid sequence shown in SEQ ID NO.1.

[0008] The application of the secretory protein FoXYG1 of Fusarium wilt of banana or related biological materials, wherein the amino acid sequence of the secretory protein FoXYG1 of Fusarium wilt of banana is shown in SEQ ID NO.1.

[0009] Furthermore, the relevant biomaterial is either biomaterial 1) or biomaterial 2):

[0010] 1) Substances that can increase the content and / or activity of FoXYG1, a protein secreted by Fusarium wilt pathogens in bananas;

[0011] 2) Substances that can inhibit or block the content and / or activity of the secreted protein FoXYG1 of Fusarium wilt in bananas.

[0012] Furthermore, the application is any one or more of the following applications A), B), C), D), and E):

[0013] A) Applications in regulating plant resistance to pathogens;

[0014] B) Applications in regulating plant immune responses;

[0015] C) Application in the prevention and control of banana wilt disease;

[0016] D) Application of cultivating plants resistant to Fusarium wilt;

[0017] E) Application of methods to regulate the pathogenicity of banana wilt pathogen.

[0018] Furthermore, the application of regulating plant resistance to pathogens includes any one or more of the following: application of biological material 1) to enhance plant resistance to pathogens and application of biological material 2) to reduce plant resistance to pathogens.

[0019] Furthermore, the pathogens include any one or more of Fusarium and Alternaria; preferably, they include any one or more of Fusarium oxysporum f.sp.cubense (Foc) race 4, Fusarium oxysporum strain Fo5176, and Alternaria alternata.

[0020] Furthermore, the application of regulating plant immune response includes any one or more of the following: application of biological materials 1) to induce / enhance plant immune response and application of biological materials 2) to inhibit / reduce plant immune response.

[0021] Furthermore, the aforementioned induction / enhancement of plant immune responses includes: inducing tobacco allergic necrosis, inducing callose accumulation, and / or upregulating marker genes of plant immune-related pathways.

[0022] Furthermore, the plants include any one or more of tobacco, Arabidopsis thaliana, and banana; preferably, any one or more of Nicotiana benthamiana, Arabidopsis thaliana Columbia, and Brazilian banana.

[0023] Furthermore, the plant immune-related pathway marker genes include any one or more of the following: salicylic acid (SA) signaling pathway marker genes NbPR1 and NbPR2, jasmonic acid (JA) signaling pathway marker genes NbPR4 and NbLOX, ethylene (ET) signaling pathway marker genes NbERF1 and NbACCO1, and allergic response (HR) signaling pathway marker gene NbHin1.

[0024] Furthermore, the application for controlling banana wilt disease is as follows: biological material 1) application for controlling banana wilt disease.

[0025] Furthermore, the application of cultivating plants resistant to wilt is as follows: 1) Application of biological materials in cultivating plants resistant to wilt.

[0026] Furthermore, the application of regulating the pathogenicity of Fusarium wilt of banana includes any one or more of the following: application of biological material 1) to reduce the pathogenicity of Fusarium wilt of banana and application of biological material 2) to increase the pathogenicity of Fusarium wilt of banana.

[0027] Furthermore, the wilt pathogens include any one or more of the following: Fusarium oxysporum f.sp.cubense (Foc) race 4 and Fusarium oxysporum strain Fo5176 (F. oxysporum, Fo5176).

[0028] Furthermore, the biological material 1) is selected from: recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the coding gene of FoXYG1.

[0029] Furthermore, the recombinant vector used is selected from the plant expression vector pBin-GFP and the pCAMBIA1302 vector.

[0030] Furthermore, the nucleotide sequence of the gene encoding FoXYG1 is shown in SEQ ID NO.2.

[0031] Furthermore, the biological material 2) is selected from: reagents that interfere with or knock out FoXYG1, and small molecule RNA inhibitors that target and inhibit the expression of the FoXYG1 encoding gene.

[0032] Furthermore, the reagents for interfering with or knocking out FoXYG1 include: a split marker homologous recombination reagent or a site-directed mutagenesis reagent targeting FoXYG1, wherein the split marker homologous recombination reagent or the site-directed mutagenesis reagent induces a loss-of-function mutation in FoXYG1, and the small RNA inhibitors that target and inhibit the expression of the gene encoding FoXYG1 include: shRNA, siRNA, miRNA, or antisense nucleic acid.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] 1. The secretory protein FoXYG1 in this invention can induce plant defense responses, including causing hypersensitive necrosis of plant cells, inducing callose accumulation and upregulating the expression of marker genes of plant immune-related pathways, and activating defense pathways mediated by SA, JA, ET and HR pathways to induce plant immune responses. It can significantly improve plant resistance to Alternaria and / or Fusarium oxysporum. It provides a new way to improve plant disease resistance and reduce disease occurrence, and therefore has broad application prospects in the green development of modern agriculture.

[0035] 2. The secretory protein FoXYG1 in this invention enhances plant disease resistance: The gene encoding the secretory protein FoXYG1 is constructed into a plant expression vector, the recombinant plasmid is transformed into Agrobacterium and transiently expressed in Tobacco Benzoentae cells, and the secretory protein FoXYG1 is transiently expressed in Tobacco Benzoentae using Agrobacterium injection technology, thereby stimulating the immune response of the plant and enhancing its resistance to Alternaria alternata. Therefore, FoXYG1 can be used as a protein that induces plant immunity in the field of plant disease control.

[0036] 3. This invention constructs a FoXYG1 gene deletion mutant by amplifying homologous fragments flanking the FoXYG1 gene of *Fusarium wilt* and simultaneously fusing it with the hygromycin phosphotransferase gene. Experiments showed that the pathogenicity of the deletion mutant was significantly enhanced compared to the wild-type strain XJZ2, a specialized strain of *Fusarium oxysporum*. In situ complementation of this gene restored its pathogenicity to the level of the wild-type strain XJZ2. The results confirm that FoXYG1 plays a negative regulatory role in the pathogenicity of *Fusarium wilt* infecting the banana host. Attached Figure Description

[0037] Figure 1 This is a diagram showing the results of an experiment on the allergic necrosis of *Nicotiana benthamiana* cells induced by the secretory protein FoXYG1.

[0038] Figure 2 This is a diagram showing the experimental results of callose accumulation in tobacco leaves induced by the secretory protein FoXYG1.

[0039] Figure 3 This is a diagram showing the experimental results of upregulation of marker genes in the immune-related pathway of Tobacco Benedict's disease induced by the secretory protein FoXYG1.

[0040] Figure 4 The figure shows the experimental results of the resistance response of tobacco to Alternaria alternifolia induced by the secretory protein FoXYG1; where A is the phenotype of Alternaria alternifolia-infected tobacco leaves; and B is the statistical analysis of the diameter of lesions on leaves infected with Alternaria alternifolia.

[0041] Figure 5 This is a diagram showing the results of an apoplast experiment on the localization of the secretory protein FoXYG1 in plants.

[0042] Figure 6 The results show the resistance of Arabidopsis thaliana overexpressing FoXYG1 to the Fusarium wilt pathogen strain Fo5176.

[0043] Figure 7 The results of pathogenicity testing of FoXYG1 knockout and complement mutants of banana wilt are shown in Figure A; A shows the infection status of wild-type strain XJZ2, FoXYG1 knockout and complement mutant strains on the roots of banana seedlings; B is a statistical graph of the pathogenicity results of each tested strain on the host banana. Detailed Implementation

[0044] The embodiments of the present invention will be described in detail below with reference to the implementation methods. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0045] Unless otherwise specified, follow standard conditions or the manufacturer's recommendations. Reagents or instruments without a manufacturer's name are all commercially available standard products.

[0046] The *Fusarium oxysporum* race 4 (Foc), strain Fo5176 (F. oxysporum, Fo5176), and *Alternaria alternata* involved in this invention are common plant pathogens that can be obtained commercially or naturally.

[0047] The strain XJZ2 of Fusarium oxysporum, a Cuban-specific physiological race, has been published in the literature “Li Minhui, Xi Pinggen, Jiang Zide, et al. Identification of physiological races of Fusarium oxysporum var. wilt in bananas in Guangdong Province [J]. Journal of South China Agricultural University, 2007, 28(2):38-41. DOI:1001-411X(2007)02-038-04.”

[0048] The strain of Fusarium oxysporum Fo5176 has been published in the literature “Fokkens L, Guo L, Dora S, et al. AChromosome-Scale Genome Assembly for the Fusarium oxysporum Strain Fo5176 To Establish a Model Arabidopsis-Fungal Pathosystem[J]. G3-Genes GenomesGenetics, 2020, 10(10): g3.401375.2020.DOI:10.1534 / g3.120.401375.”

[0049] Alternaria alternata has been published in the literature “Fang Yuda, Liu Dajun. Transgenic tobacco plants with rice chitinase gene and their resistance to tobacco red spot disease (Alternaria alternata) [J]. Journal of Nanjing Agricultural University, 2000, 23(1):5.DOI:10.3321 / j.issn:1000-2030.2000.01.002..”

[0050] The Escherichia coli DH5α competent cells and Agrobacterium GV3101 involved in the embodiments of the present invention can be obtained through conventional commercial means.

[0051] The pBin-GFP vector and pCAMBIA1302 vector involved in the embodiments of this invention can be obtained through conventional commercial purchases;

[0052] The pDAN vector was donated by Professor Shaobin Zhong of North Dakota State University and has been published in the paper “Li M, Xie X, Lin X, et al. Functional characterization of the gene FoOCH1 encoding a putative α-1,6-mannosyltransferase in Fusarium oxysporum f.sp.cubense. Fungal Genetics and Biology. 2014, 65:1-13. DOI:10.1016 / j.fgb.2014.01.005.”.

[0053] The pKNT-G418 vector was donated by Associate Professor Yun Yingzi of Fujian Agriculture and Forestry University and has been published in the literature "Li M, Xie L, Wang M, et al. FoQDE2-dependent milRNA promotes Fusarium oxysporum f.sp.cubensevirulence by silencing a glycosyl hydrolase coding gene expression. PLoSPathogens, 2022, 18(5):e1010157. DOI:10.1371 / journal.ppat.1010157".

[0054] Example 1: Cloning and vector construction of the FoXYG1 gene

[0055] Design full-length specific primers for FoXYG1, containing homologous sequences at both ends of the vector cloning site, including:

[0056] Forward primer FoXYG1-F1:

[0057]

[0058] Reverse primer FoXYG1-R2:

[0059]

[0060] The thickened sequence is a sequence that is completely identical to the two ends of the vector pBin-GFP, which facilitates subsequent homologous recombination.

[0061] Forward primer FoXYG1-F2:

[0062]

[0063] Reverse primer FoXYG1-R2:

[0064]

[0065] The bold sequence is a sequence that is completely consistent with the two ends of the vector pCAMBIA1302, which facilitates subsequent homologous recombination.

[0066] Genomic RNA of Fusarium oxysporum was extracted using the Trizol method to obtain Total RNA, and cDNA was synthesized using the TAKARA PrimeScript™ RT Master Mix (Perfect Real Time) reverse transcription kit.

[0067] Novozyme high-fidelity enzyme ( Turbo Super-Fidelity DNA Polymerase was used to amplify the full length of FoXYG1 using cDNA of Fusarium wilt pathogen race 4 as a template. The specific PCR system (50 μl) consisted of: 1 μl cDNA template, 2 μl each of 10 μmol / L primers FoXYG1-F and FoXYG1-R, 1 μl dNTP, 25 μl 2× Buffer, 1 μl Phanta, and 18 μl ddH2O.

[0068] The PCR reaction program was as follows: pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 15 s, annealing at 60℃ for 15 s, extension at 72℃ for 45 s, and final extension at 72℃ for 5 min, for 34 cycles.

[0069] PCR products were recovered from the agarose gel by a DNA recovery kit (OMEGA) after agarose gel electrophoresis, followed by ClonExnase. TM II. The One Step Cloning Kit (Vazyme) was used to digest the SmaⅠ site of the plant expression vector pBin-GFP and the Nco1 site of the pCAMBIA1302 vector, respectively, and the fragments were recovered using the above method. Each fragment was then ligated to the target fragment. The obtained recombinant plasmids pBin-GFP::FoXYG1 and pCAMBIA1302::FoXYG1 were transformed into *E. coli* DH5α competent cells and screened on LB agar containing 100 μg / ml kanamycin. Plasmids were extracted from the strain using the OMEGA plasmid extraction kit and sequenced by Shanghai Sangon Biotech. The open reading frame of FoXYG1 was finally determined, and its nucleic acid sequence is shown in SEQ ID NO.2. The amino acid sequence of its encoded protein is shown in SEQ ID NO.1.

[0070] FoXYG1 amino acid sequence (SEQ ID NO.1):

[0071] MHIASYVSVAFTVLGLAAASPKGCTTKRSPEPKLENGHYIVDRATKFANKKVWTFNGKSLPEGLYSSDYPVGKTHVFTPSGVKVRNGYLELTVPGGQKSKPYKAAEVATEIENIKYASVRTTAILSEPAGVC NGMFFYQSDSQETDIEWLSDPKSESNYDGIRRLWFTNQDNDGDGEPSHKPVLPPSNPTTTEHEYRIDWTKGLVQFYVDGVKQWSTKKDVPNVPGPWIWNNWSNGDKGWSAGPPKENAVFKIKKIEMYYNTA;

[0072] FoXYG1 nucleic acid sequence (SEQ ID NO.2):

[0073]

[0074] Example 2: Transient expression of FoXYG1 in Nicotiana benthamiana induces hypersensitive necrosis of cells.

[0075] The recombinant plasmid pBin-GFP::FoXYG1 obtained in Example 1 was transformed into 100 μl of Agrobacterium GV3101 competent cells. Single colonies of Agrobacterium GV3101 with pBin-GFP and single colonies of Agrobacterium GV3101 transfected with the INF1 plasmid (GenBank: AY830094.1) were obtained using the same method. Screening was performed using LB solid medium containing 100 μg / ml kanamycin. After single colonies were verified as correct by colony PCR, they were cultured in liquid LB medium with shaking at 28°C and 180 rpm for 1-2 days.

[0076] Centrifuge the above three bacterial cells at 4000 rpm for 4 min, discard the supernatant, resuspend the bacterial cells in 10 mmol / L MgCl2, and wash 3-4 times. OD 600 Adjust the concentration to 0.6-1.0. Using a sterile syringe (1 ml), inject Agrobacterium suspension into the underside of vigorously growing 5-6 leaf-stage *Nicotiana benthamiana* plants to transiently express the target gene in *Nicotiana benthamiana* cells. Each leaf was injected with pBin-GFP Agrobacterium as a negative control and pBin-GFP::INF1 Agrobacterium as a positive control. The injected tobacco plants were cultured in a greenhouse (22℃, 16h light / 8h dark). The experiment was repeated three times. Five days after Agrobacterium injection and expression, leaf necrosis was observed. Once a clear phenotype appeared, leaves were collected and photographed.

[0077] Results: Obvious hypersensitive necrosis was observed in leaves injected with Agrobacterium containing the pBin-GFP::INF1 recombinant vector, while a weak hypersensitive necrosis reaction was observed in areas injected with Agrobacterium containing pBin-GFP::FoXYG1. The negative control pBin-GFP showed no necrosis. This indicates that FoXYG1 can induce hypersensitive necrosis in leaves of Nicotiana benthamiana. Figure 1 ).

[0078] Example 3: FoXYG1 induces callose accumulation in tobacco leaves

[0079] After expressing pBin-GFP::FoXYG1 and pBin-GFP in Tobacco Benedict's leaves for 36 hours as described in Example 2, the leaves were collected and immersed in freshly prepared aniline blue staining solution (0.1 mg / ml aniline blue, 0.1 M NaH2 PO4, pH adjusted to 9) with the leaf back facing down. After standing in the dark for 1-2 hours, the leaves could be observed and photographed under a microscope.

[0080] Results: Compared to the negative control, leaves injected with Agrobacterium containing the pBin-GFP::FoXYG1 recombinant vector showed significant callose accumulation. Figure 2 The results showed that FoXYG1 could induce callose accumulation in tobacco leaves.

[0081] Example 4: FoXYG1 induces upregulation of marker genes in the immune-related pathway of Tobacco Benzoenterae.

[0082] After expressing the above three bacterial strains in *Nicotiana benthamiana* leaves for 36 hours as described in Example 2, leaves were collected, and RNA was extracted according to the instructions of the All-In-One RNA Mini-Preps Kit (BBI). RNA was then extracted using PrimeScript. TM The RTreagent kit with gDNA Eraser (Takara) was used to reverse transcribe cDNA. An appropriate amount of the reverse transcription product was then used for RT-qPCR to detect the expression of immune-related pathway marker genes (NbPR1, NbPR2, NbPR4, NbLOX, NbERF1, NbACCO1, NbHin1). FOIG-08405, belonging to the GH16 family of Foc, was transiently expressed in tobacco using the same method, and the expression of marker genes was detected by RT-qPCR. The primer sequences for real-time quantitative PCR are as follows:

[0083] qNbPR1-F:5'_CCGCCTTCCCTCAACTCAAC_3'

[0084] qNbPR1-R:5'_GCACAACCAAGACGTACTGAG_3'

[0085] qNbPR2-F: 5'_AGGTGTTTGCTATGGAATGC_3'

[0086] qNbPR2-R:5'_TCTGTACCCACCATTCTGC_3'

[0087] qNbPR4-F: 5'_GGCCAAGATTCCTGTGGTAGAT_3'

[0088] qNbPR4-R: 5'_CACTGTTGTTTGAGTTCCTGTTCCT_3'

[0089] qNbLOX-F:5'_AAAACCTATGCCTCAAGAAC_3'

[0090] qNbLOX-R:5'_ACTGCTGCATAGGCTTTGG_3'

[0091] qNbERF1-F:5'_GCTCTTAACGTCGGATGGTC_3'

[0092] qNbERF1-R:5'_AGCCAAACCCTAGCTCCATT_3'

[0093] qNbACCO1-F:5'_GTGGCCAGTAACGGTCTTGA_3'

[0094] qNbACCO1-R:5'_TCCGAGTCCCGTCTTTTTGG_3'

[0095] qNbHin1-F:5'_ATGCCACGTCATAATCCCGT_3'

[0096] qNbHin1-R:5'_AATCTTCAACGTCCTATGACCTGA_3'

[0097] Real-time quantitative PCR reaction:

[0098] PCR reaction system (20 μl): 10 μl of 2×ChamQ Universal SYBR qPCR Master Mix (Vazyme), 100 ng of cDNA, 0.4 μl each of upstream and downstream primers (10 μm), and ddH2O added to 20 μl.

[0099] Reaction program: I: 95℃ for 30s; II: 95℃ for 30s, 60℃ for 30s; Program II for 40 cycles. The melting curve analysis program is: 95℃ for 15s, 60℃ for 1min, 95℃ for 15s.

[0100] Results: After 36 h of transient expression of FoXYG1 in tobacco leaves, the expression of salicylic acid (SA) signaling pathway markers NbPR1 and NbPR2, jasmonic acid (JA) signaling pathway markers NbPR4 and NbLOX, ethylene (ET) signaling pathway markers NbERF1 and NbACCO1, and anaphylactic response (HR) signaling pathway marker NbHin1 were all significantly upregulated compared with control leaves. Figure 3 This indicates that FoXYG1 can effectively induce the upregulation of marker genes in the immune-related pathway of Nicotiana benthamiana. Notably, FOIG-08405, also a member of the GH16 family, did not induce the upregulation of these marker genes under the same conditions, showing no significant difference from the control leaves. This suggests that FoXYG1 induces a specific immune response in plants.

[0101] FOIG-08405 amino acid sequence (SEQ ID NO.3):

[0102] MKSLFLTLMVLTATVASPVSKITPLIKPGFAPTFIDTFFGYPGSLPSSSNWIFDLGTSYPGGAERWGNNEFETYTKDPSNVHITKDQNLAITPRLKEGKWTSARIETQRSDFVAKEGGKLLVEARLKVGGAPASQMQGIWPAFWALGTEFR GNYTNWPMATEWDILEVINGESKMYSTIHCGTAPGGPCNEYNGIGSGGRDFSRGEFHTLGFMVDRSMCGEGKNGSWRDESLNWFLDGKKIFNVTGATVGDEPTWVKLAHKEHFLLLNVAVGGNWPGPPNNATIDGPSVNLEVDYVGVWNSL.

[0103] Example 5: Secretory protein FoXYG1 induces resistance in Nicotiana benthamiana to Alternaria alternata.

[0104] The recombinant plasmid pBin-GFP::FoXYG1 was transformed into Agrobacterium GV3101 according to the method in Example 2. After centrifugation of the bacterial cells, the bacterial cells were suspended in 10 mmol / L MgCl2, and the OD was... 600 Adjust the concentration to 0.6–1.0. Using a sterile syringe (1 ml), inject Agrobacterium suspension into the underside of vigorous 5-6 leaf-stage *Nicotiana benthamiana* leaves. Inject Agrobacterium containing the pBin-GFP::FoXYG1 recombinant vector into the left half of the same leaf, and pBin-GFP Agrobacterium as a control into the right half. 36 hours after injection, cut the injected leaves and place them on moist filter paper. Using a sterile 5 mm perforator, collect mycelial cakes of uniform age from the edge of *Alternaria alternata* colonies. Inoculate one mycelial cake on each side of the underside of the leaf, repeating this process for 20 leaves. Incubate at 25°C for 5 days, then photograph and measure the diameter of the lesions on both sides.

[0105] Results: Through observation and biological statistics, the diameter of Alternaria alternata lesions in leaves of Nicotiana benthamiana expressing FoXYG1 was significantly smaller than that in the control (GFP). Figure 4 This indicates that expression of the secretory protein FoXYG1 in tobacco can induce plant resistance and inhibit Alternaria alternata infection.

[0106] Example 6: Secretory protein FoXYG1 is located in the apoplast of plants.

[0107] After expressing the transformed pBin-GFP::FoXYG1 and pBin-GFP Agrobacterium GV3101 in Tobacco Benedict's leaves for 36 hours as described in Example 2, the leaves were collected and treated with 0.7M mannitol to obtain plasmolysis samples. The fluorescence of tobacco cells was then observed and photographed using a laser confocal microscope (Nikon A1).

[0108] Results: The results showed that FoXYG1 was localized only in the periphery of Nicotiana benthamiana cells, while the pBin-GFP empty vector control was distributed both in the periphery and on the nucleus. After plasmolysis, FoXYG1 fluorescence was observed in the aplastic space, while the fluorescence of the pBin-GFP empty vector was only observed inside the cell membrane. Figure 5 The results showed that FoXYG1 is located in the apoplast of plants and is a secretory protein.

[0109] Example 7: Genetic transformation in Arabidopsis thaliana

[0110] First, competent Agrobacterium GV3101 cells were prepared, and then the positive pCAMBIA1302::FoXYG1 cloning plasmid was transformed into the competent Agrobacterium GV3101 cells. Arabidopsis thaliana Colombian wild-type Col-0 was cultured using tissue culture. After the cells developed four leaves and a central bud, they were transplanted for soil culture. Once inflorescences appeared, the competent Agrobacterium GV3101 cells were used to infect and transform Arabidopsis thaliana. Using the Kan resistance site on the pCAMBIA1302 vector, appropriate concentrations of kanamycin and hygromycin eukaryotic resistance were used for selection. To prevent false positives, genomic DNA was extracted from leaves of suspected positive T1 plants. PCR detection was used to screen for Arabidopsis thaliana overexpressing the FoXYG1 gene. Total RNA was extracted, reverse transcribed into cDNA, and qRT-PCR was used to detect the expression level of the FoXYG1 gene in the transgenic Arabidopsis plants, confirming the existence of transgenic Arabidopsis thaliana overexpressing FoXYG1. The same method was used to screen for T3 generation homozygous positive transgenic Arabidopsis thaliana lines.

[0111] Example 8: Determination of resistance to Fusarium wilt in Arabidopsis thaliana overexpressing FoXYG1

[0112] Fusarium oxysporum strain Fo5176 was cultured in 200 ml of YPD liquid medium with shaking (25℃, 180 rpm) for 2 days. Spores were collected by high-speed centrifugation after filtration through filter paper, and the spore concentration was adjusted to 1×10⁻⁶ with sterile water. 5Approximately 100 spores per ml were used for inoculation experiments. Wild-type Arabidopsis thaliana Col-0 and the mutant FoXYG1 strains of uniform growth, aged 2-3 weeks, were selected for inoculation with strain Fo5176. Sterile water was used as a negative control, and the inoculation was repeated 20 times. Specific procedures: The roots of the Arabidopsis thaliana plants were rinsed thoroughly with tap water, soaked in the spore suspension for 1 minute, and then transplanted back into pots, followed by irrigation with an appropriate amount of spore suspension. The inoculated plants were placed in a greenhouse for cultivation, watered every 3-4 days during the day (16h, 22℃) and night (8h, 20℃). Disease incidence was observed after 3-4 weeks.

[0113] Results: After 3-4 weeks, the wild-type Col-0 strain inoculated with Fo5176 showed stunted and deformed plants with wrinkled leaves, slow growth, and weak resistance to pathogen infection. Compared with the wild-type Col-0 strain, the mutant FoXYG1 strain showed significantly weaker disease incidence and exhibited some resistance to pathogen infection. Figure 6 The results showed that FoXYG1 can act as an effector to activate the immune response in plants.

[0114] Example 9: Knockout of FoXYG1, the pathogen of Fusarium wilt in bananas

[0115] The split marker homologous recombination method was used to amplify the homologous fragments flanking FoXYG1 and fuse them with the hygromycin gene to construct a homologous fragment containing the resistance gene to replace the target fragment and achieve the knockout of the target gene.

[0116] First round of PCR amplification: using specific primers (containing fusion adapters), Novizan high-fidelity enzyme (… Turbo Super-Fidelity DNA Polymerase amplified fragments of the left and right arms (LB, RB) of FoXYG1 and the left and right arms (HPH-up+, HPH-down) of the hygromycin gene using wild-type strain DNA and pDAN plasmid as templates, respectively. The systems are shown in Table 1.

[0117] Table 1 PCR reaction system

[0118]

[0119] The procedure is as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 15 s, annealing at 56-72℃ for 15 s, extension at 72℃ for 30-60 s / kb, 30 cycles, and finally extension for 5 min.

[0120] Second round of PCR amplification (fusion): Purified PCR products were obtained through gel extraction or PCR product purification methods. Using these products as templates, fusion PCR was performed on DNA from LB+HPH-up and HPH-down+RB, respectively. The amplification system and procedure are as described above.

[0121] Preparation of protoplasts

[0122] (1) Take the wild-type strain XJZ2 that has been grown at a constant temperature of 28℃ for 7 days on a PDA plate, wash the plate with an appropriate amount of sterile water, and filter the mycelium into a 50ml centrifuge tube using sterile three-layer lens paper.

[0123] (2) Place the 50m centrifuge tube in a centrifuge at 4℃ and centrifuge at 4000rpm for 10min.

[0124] (3) Discard the supernatant, add 1 ml of YPD to a 50 ml centrifuge tube, transfer the precipitate to an Erlenmeyer flask containing 150 ml of YPD, place it in a shaker at 28 ℃ and shake at 180 rpm for 8 h.

[0125] (4) Take an appropriate amount of the above bacterial solution into a 50ml centrifuge tube and centrifuge at 3000rpm for 5min in a centrifuge at 4℃.

[0126] (5) Discard the supernatant, add 50 ml of 0.8 M NaCl to wash the precipitate, and centrifuge at 3000 rpm for 5 min at 4℃.

[0127] (6) Add 5 ml of enzyme hydrolysate to suspend the precipitate, place it in a shaker at 32℃ and shake at 120 rpm for 4 h.

[0128] (7) Add 0.8M NaCl to the centrifuge tube to 50ml and centrifuge at 3000rpm for 5min at 4℃.

[0129] (8) Discard the supernatant, add 50 ml of STC solution to suspend the precipitate, and centrifuge at 3000 rpm for 5 min in a centrifuge at 4℃.

[0130] (9) Add 800 μl STC, 200 μl PTC and 20 μl DMSO to suspend the precipitate, and the concentration of protoplasts should be at least 10. 7 Mix the samples at a concentration of 1 / ml, then aliquot them into sterile 1.5ml centrifuge tubes and store at -80℃.

[0131] PEG-mediated protoplast transformation

[0132] (1) In a clean bench, add 100 μl of protoplasts to a sterile 50 ml centrifuge tube and pre-cool it on ice.

[0133] (2) Add 20 μl of plasmid DNA to a 50 ml centrifuge tube containing protoplasts after pre-cooling, mix gently, and incubate on ice for 30 min.

[0134] (3) Prepare a 50% PEG solution in advance during the ice bath process.

[0135] (4) Gently add 100 μl, 300 μl and 600 μl of 50% PEG solution to the above 50 ml centrifuge tubes respectively. The operation should not be violent.

[0136] (5) Let stand at room temperature for 20 minutes, then gently add 1 ml, 3 ml and 6 ml of STC solution to the centrifuge tubes respectively.

[0137] (6) Centrifuge at 3000 rpm for 10 min at 4℃.

[0138] (7) Discard the supernatant until about 400 μl of liquid remains. Mix the remaining liquid with 1.6 ml of RM medium and place it in a shaker at 26°C for 3 h at 60 rpm.

[0139] (8) Dissolve the RMA medium in advance and cool it to about 50°C. Add the RMA medium to the 50ml centrifuge tube containing protoplasts to 50ml, and add 50μl of hygromycin (50μg / ml). Mix well, invert the plate, and then incubate it in an incubator at 28°C for 2 days.

[0140] Screening and validation of FoXYG1 gene knockout mutants

[0141] (1) Resistance plate screening: After culturing in RMA regeneration medium containing hygromycin (50 μg / ml) for 2 days, single colonies of the transformed strains were selected and transferred to new hygromycin-resistant plates for re-screening. The plates were then incubated at 28°C. After 3 days of growth, DNA was extracted from transformants that still grew normally on the resistance plates using a fungal DNA extraction kit (OMEGA) for further verification.

[0142] (2) PCR identification: Based on the target fragment sequence, a pair of inner-arm primers were designed to verify the successful introduction of the resistance gene into the homologous region, and a pair of outer-arm primers were designed to verify the in situ substitution of the resistance gene. The obtained transformants were amplified by PCR using the two pairs of specific verification primers. Transformants with band sizes that met the expectations were screened and cultured for further qRT-PCR verification of the target fragment.

[0143] (3) qRT-PCR verification: Total RNA was extracted from the transformants and reverse transcribed to obtain cDNA. The cDNA of the transformed transformants was detected by real-time quantitative PCR using ChamQ Universal SYBR qPCR Master Mix (Vazyme). Based on the difference in expression levels, the correct knockout mutant (ΔFoXYG1) was screened out.

[0144] Example 10: Replenishment of FoXYG1, the pathogen of banana wilt

[0145] In situ complementation of FoXYG1 knockout mutants was performed using the split marker homologous recombination method.

[0146] First round of PCR amplification: Using DNA from the wild-type strain XJZ2 of *Fusarium oxysporum* (Cuba specialized type) as a template, primers were designed to amplify the FoXYG1 fragment and its left and right arms (LB, RB). Simultaneously, using the pKNT-G418 plasmid as a template, primers were designed to amplify the left and right arms (G418-up, G418-down) of the G418 resistance gene. The amplification system and procedure were the same as above.

[0147] Second round of PCR amplification (fusion): The PCR products from the previous step were excised and recovered to obtain individual DNA fragments. The DNA fragments LB+FoXYG1+G418-up and G418-down+RB were fused separately to obtain dual DNA fragments for in situ complementation. The amplification system and procedure were the same as above.

[0148] By preparing protoplasts of the FoXYG1 knockout mutant and conducting PEG-mediated protoplast transformation experiments, the grown complement transformants were screened and verified using G418 resistance, and finally the correct FoXYG1 complement mutant (CO-FoXYG1) was selected. The specific operation was the same as in Example 10.

[0149] Example 11: The effect of knockout and reintroduction of FoXYG1, the causal agent of banana wilt, on its pathogenicity.

[0150] (1) Select healthy Brazilian banana (AAA) tissue culture seedlings with 5-6 leaves of similar growth for pathogenicity testing. Dilute the spores of the test strain to a concentration of 10. 7 After properly cleaning the roots, the banana seedlings were soaked in a spore suspension for 30 minutes, then planted in sterilized sand. Water was used as a control. Thirty banana seedlings were inoculated for each treatment and cultured in a 28℃ greenhouse. After 3-4 weeks, the base of the pseudostem was cut to observe the degree of browning of the vascular bundles, and the incidence of disease was observed and recorded.

[0151] (2) The disease incidence of banana seedlings after inoculation was graded and statistically analyzed. The grading criteria for the disease incidence at the base of the pseudostem of banana tissue culture seedlings are shown in Table 2:

[0152] Table 2 Grading Standards for Disease Symptoms at the Base of Banana Seedling Pseudostem

[0153]

[0154]

[0155] Results: Bananas inoculated with ΔFoXYG1 showed more severe symptoms in the roots and leaves, with most plants exhibiting yellowing and wilting of leaves, and more than half of the root vascular bundles turning brown. The control group (CK) showed no symptoms. Similarly, the proportion of secondary and tertiary disease in ΔFoXYG1 was higher than that in wild-type XJZ2 and CO-FoXYG1. Figure 7 The results showed that the absence of the FoXYG1 gene significantly increased the pathogenicity of Fusarium wilt in bananas, indicating that the FoXYG1 gene plays a negative regulatory role in the pathogenic process of Fusarium wilt infecting the host plant, banana. Combined with previous experimental results (the secretory protein encoded by the FoXYG1 gene can activate the plant's immune response), it is clear that if the pathogen does not produce this secretory protein, it cannot activate the plant's immune response, thus enabling the pathogen to successfully infect the host plant. Therefore, the FoXYG1 secretory protein can act as an effector to activate the plant's immune response.

[0156] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of FoXYG1 secreted protein of Fusarium wilt of banana or related biological materials, characterized in that: The amino acid sequence of the secretory protein FoXYG1 of the banana wilt pathogen is shown in SEQ ID NO.

1. The relevant biological materials are selected from: recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the coding gene of FoXYG1. The application mentioned is any one or more of the following applications: A), B), C), D), and E): A) Application of overexpressing the gene encoding the secretory protein FoXYG1 of Fusarium wilt in bananas to improve plant resistance to the pathogen; B) Application of overexpression of the gene encoding the secretory protein FoXYG1 of Fusarium wilt in bananas to induce / enhance plant immune responses; C) Application of overexpression of the gene encoding the secretory protein FoXYG1 of Fusarium wilt in the prevention and control of Fusarium wilt in bananas; D) Application of overexpressing the gene encoding the secretory protein FoXYG1 of banana wilt pathogen in the cultivation of wilt-resistant plants; E) Application of overexpressing the gene encoding the secretory protein FoXYG1 of Fusarium wilt in reducing the pathogenicity of Fusarium wilt in bananas; The pathogens mentioned are any one or more of Fusarium and Alternaria. The plant mentioned is any one or more of tobacco, Arabidopsis thaliana, and banana; The aforementioned induction / enhancement of plant immune responses includes: inducing allergic necrosis in tobacco, inducing callose accumulation, and / or upregulating marker genes of plant immune-related pathways.

2. The application of FoXYG1 secreted protein of Fusarium wilt of banana or related biological materials, characterized in that: The amino acid sequence of the secreted protein FoXYG1 of the banana wilt pathogen is shown in SEQ ID NO.

1. The relevant biological material is selected from: Split marker homologous recombination reagent or site-directed mutagenesis reagent targeting FoXYG1, wherein the Split marker homologous recombination reagent or site-directed mutagenesis reagent performs loss-of-function mutation on FoXYG1. The application is any one or more of the following applications I), II), and III): I) Knocking out the gene encoding the secretory protein FoXYG1 of the banana wilt pathogen reduces the plant's resistance to the pathogen; II) Application of knocking out the gene encoding the secretory protein FoXYG1 of banana wilt pathogen to suppress / reduce plant immune response; III) Application of knocking out the gene encoding the secretory protein FoXYG1 of Fusarium wilt in bananas to enhance the pathogenicity of Fusarium wilt in bananas; The pathogens mentioned are any one or more of Fusarium and Alternaria. The plant in question is any one or more of tobacco, Arabidopsis thaliana, and banana.

3. The application according to claim 1 or 2, characterized in that: The pathogen mentioned is: Fusarium oxysporum, Cuban variant (… Fusarium oxysporum f. sp. cubense , Foc )4 physiological race, Fusarium oxysporum strain Fo5176 ( F. oxysporum Fo5176) and Alternaria alternifolia ( Alternaria alternata Any one or more of the following; The plant mentioned is any one or more of Nicotiana benthamiana, Arabidopsis thaliana Columbia type, and Banana brasiliensis; The aforementioned plant immune-related pathway marker gene is: salicylic acid signaling pathway marker gene. NbPR1, NbPR2 Marker genes of the jasmonic acid signaling pathway NbPR4, NbLOX Marker genes of the ethylene signaling pathway NbERF1, NbACCO1 and marker genes of allergic reaction signaling pathway NbHin1 Any one or more of the following.

4. The application according to claim 1 or 2, characterized in that: The fungus causing banana wilt is: Fusarium oxysporum, Cuban variant (… Fusarium oxysporum f. sp. cubense , Foc )4 physiological races and Fusarium oxysporum strain Fo5176 ( F. oxysporum Any one or more of the following: Fo5176.

5. The application according to claim 1, characterized in that: The recombinant vector used was selected from the plant expression vector pBin-GFP and the pCAMBIA1302 vector; The nucleotide sequence of the gene encoding FoXYG1 is shown in SEQ ID NO. 2.

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