Application of VdR4 gene in improving cotton resistance to verticillium wilt

Abstract

The application discloses application of a VdR4 gene in improving cotton verticillium wilt resistance and belongs to the field of biotechnology application.The VdR4 gene disclosed by the application encodes beta,1,3-glucanase.The application provides a full-length ORF nucleotide sequence and an amino acid sequence of VdR4 in a heterologous tetraploid island cotton material H7124.Cotton VdR4 interacts with an effector protein PeVD1 of a verticillium wilt fungus and participates in cotton verticillium wilt resistance.VdR4 is significantly up-regulated in expression in cotton roots induced by the verticillium wilt fungus.Overexpression of the gene in cotton can significantly improve the disease resistance of plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to the application of the cotton VdR4 gene in improving cotton resistance to Verticillium wilt. Transcriptome analysis of roots of island cotton H7124 induced by Verticillium wilt fungus V991 identified a gene, VdR4, which is significantly upregulated by the fungus and encodes β,1,3-glucanase. RT-qPCR analysis showed that the expression of this gene in both island and upland cotton materials was induced by Verticillium wilt. In vitro antibacterial assays showed that VdR4 has an in vitro antibacterial effect against Verticillium wilt. Subcellular localization showed that VdR4 is located in the cell membrane. Further research showed that VdR4 can be secreted into the apoplast after salicylic acid induction. The full-length ORF sequence and encoded amino acid sequence of VdR4 were obtained from island cotton H7124 using PCR technology. The specific mechanisms by which this gene participates in plant disease resistance were studied using biotechnology, and overexpression of this gene in cotton significantly improved plant disease resistance. By using VdR4 as a bait protein to screen interacting proteins in a yeast expression library of Verticillium wilt pathogen V991 induced by roots of H7124 cotton (Sea Island cotton), further full-length gene cloning and interaction verification were conducted. Combined with transgenic verification and molecular mechanism analysis, it was clarified that VdR4 interacts with the Verticillium wilt pathogen toxic protein PeVD1 in cotton and participates in cotton Verticillium wilt resistance. Previous studies have reported that PeVD1 is an important effector factor in Verticillium wilt, which can target the cell nucleus and cause cytotoxicity. Our study clarified that PeVD1 is located in the cell membrane and nucleus. When PeVD1 and VdR4 are co-expressed in tobacco, PeVD1 and VdR4 exhibit apoplast localization, indicating that VdR4 in the apoplast prevents PeVD1 from targeting the cell nucleus. Overexpression of VdR4 in cotton significantly improved cotton resistance to Verticillium wilt, and a new cotton germplasm with high resistance to Verticillium wilt was obtained. Background Technology

[0002] Cotton Verticillium wilt is a significant disease in cotton production and is also one of the targets of national agricultural plant quarantine. Caused by the soil-borne fungus *Verticillium dahliae*, it is a severe vascular disease. Infected cotton plants suffer from chlorosis, turning yellow and wilting, eventually leading to plant death and severely reducing yield and fiber quality. Chemical control strategies are not ideal for Verticillium wilt, and management practices such as crop rotation can only moderately alleviate its occurrence. Identifying key disease-resistant genes in cotton and creating resistant materials is the most direct and effective strategy to improve cotton's resistance to Verticillium wilt.

[0003] Identifying resistance sources has successfully improved the resistance of other crop varieties to Verticillium wilt. Diwan et al. (1999) found that in tomatoes, resistance to Verticillium dahliae race 1 is determined by a dominant gene VE. Among the four major cultivated cotton varieties, island cotton exhibits significantly higher resistance to Verticillium dahliae infection than the most widely planted upland cotton variety in the world. However, compared to upland cotton, island cotton has lower yields, smaller planting area, and lower economic benefits. Therefore, researchers can discover key genes for Verticillium wilt resistance in island cotton, thereby cultivating superior transgenic upland cotton varieties resistant to Verticillium wilt through molecular breeding. Zhang et al. (2007) cloned a tomato VE homolog, GbVE1, from an island cotton variety resistant to Verticillium wilt. The study found that the GbVE1 gene is induced by Verticillium dahliae and the plant hormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. After cotton was inoculated with Verticillium wilt, the resistant island cotton showed a faster induction rate for GbVE1, with a much stronger induction effect than the susceptible upland cotton. Transforming Arabidopsis thaliana and upland cotton with the GbVE1 gene via Agrobacterium-mediated transformation resulted in increased resistance to Verticillium wilt in the transgenic Arabidopsis thaliana and upland cotton.

[0004] The life cycle of Verticillium wilt begins with the germination of microsclerotia. Upon sensing secretions released from the plant roots, the microsclerotia begin to produce germ tubes, which extend longitudinally along the root epidermal cells. A small number of hyphae swell at their tips, attaching themselves tightly to the root surface as appendages, forming narrow infection nails that penetrate the epidermis for infection. During infection, the pathogen secretes a gelatinous substance to firmly fix its appendages to the infection interface, absorbing the recoil force generated during penetration. Simultaneously, it releases large amounts of cell wall hydrolases, such as pectinase, cutinase, and cellulase, which damage plant cell wall tissues. Numerous effector factors disrupt or interfere with the plant's immune response. After successful invasion, the hyphae gradually spread towards the center, eventually entering the xylem and colonizing, growing and multiplying within or between cells, and extending upwards along the xylem vessels to the stem or leaves.

[0005] Furthermore, certain effector proteins secreted by *Verticillium wilt* are also important accomplices in its infection of hosts. Similar to other fungal pathogens, the *Verticillium wilt* genome encodes over 700 secreted proteins. Wang et al. (2020) systematically studied the transiently expressed predictable fungal effector proteins (including 123 scps) in strain V991, identifying three effectors that lead to cell death and demonstrating that they require BAK1 and SOBIR1 to trigger host immunity. Kombrink et al. (2017) investigated LysM effectors in *Verticillium wilt*. Through comparative genomics, they identified three core LysM effectors preserved in a collection of *Verticillium wilt* strains. However, they demonstrated that these three effectors did not contribute to virulence across different host taxa. Interestingly, they discovered a lineage-specific LysM effector in *Verticillium wilt* VdLs17, which is virulent to tomato but not to Arabidopsis thaliana. Zhang et al. (2017) discovered an effector protein (VdSCP7) in Verticillium wilt strain V592 that can target the plant cell nucleus and regulate immunity to fungal infection.

[0006] In summary, the screening of genes related to cotton Verticillium wilt resistance provides theoretical guidance and genetic resources for improving cotton disease resistance through molecular design breeding or genetic engineering. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the cotton VdR4 gene in improving cotton disease resistance or cultivating new cotton germplasm with enhanced disease resistance. Using cotton materials transgenic with the VdR4 gene, it was demonstrated that overexpression of VdR4 significantly improves plant resistance to Verticillium wilt. Using this gene as a target gene, through genetic engineering methods such as transgenics, the VdR4 gene was overexpressed in plants, resulting in the cultivation of new cotton germplasm with normal growth and development, no significant difference in cotton fiber yield and quality compared to the control, and significantly improved resistance, which was then applied in production.

[0008] Another object of the present invention is to provide a method for improving the resistance of cotton to Verticillium wilt.

[0009] Another objective of this invention is to provide a cotton VdR4 gene and its encoded protein, and to provide the full-length cDNA ORF nucleotide sequence of the gene and the amino acid sequence of the encoded protein in the island cotton material H7124.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention seeks to protect the use of the VdR4 gene, a substance that promotes the expression of the VdR4 gene, or a substance that increases the activity or content of the protein encoded by the VdR4 gene in the following a1) or a2):

[0012] al) Improve cotton's disease resistance;

[0013] a2) Develop new cotton germplasm with improved disease resistance;

[0014] The VdR4 gene is a DNA molecule having the nucleotide sequence shown in SEQ ID NO.1.

[0015] Furthermore, in the above applications, the substance that promotes the expression of the VdR4 gene or increases the activity or content of the protein encoded by the VdR4 gene is a biological material, and the biological material is any one of the following b1) to b6):

[0016] b1) An expression cassette containing the VdR4 gene;

[0017] b2) A recombinant vector containing the VdR4 gene, or a recombinant vector containing the expression cassette described in b1);

[0018] b3) Recombinant microorganisms containing the VdR4 gene, or recombinant microorganisms containing the expression cassette described in b1), or recombinant microorganisms containing the recombinant vector described in b2);

[0019] b4) A transgenic plant cell line containing the VdR4 gene, or a transgenic plant cell line containing the expression cassette described in b1), or a transgenic plant cell line containing the recombinant vector described in b2);

[0020] b5) Transgenic plant tissue containing the VdR4 gene, or transgenic plant tissue containing the expression cassette described in b1), or transgenic plant tissue containing the recombinant vector described in b2);

[0021] b6) A transgenic plant organ containing the VdR4 gene, or a transgenic plant organ containing the expression cassette described in b1), or a transgenic plant organ containing the recombinant vector described in b2).

[0022] Furthermore, in the above applications, the protein encoded by the VdR4 gene is a protein having the amino acid sequence shown in SEQ ID NO.2.

[0023] Furthermore, using the VdR4 gene as a target gene, the VdR4 gene is overexpressed through genetic engineering methods to improve cotton disease resistance or to cultivate new cotton germplasm with improved disease resistance.

[0024] Furthermore, the disease resistance mentioned refers to resistance to Verticillium wilt.

[0025] Secondly, the present invention claims protection for a method for improving the resistance of cotton to Verticillium wilt by overexpressing the VdR4 gene in cotton; wherein the VdR4 gene is a DNA molecule having the nucleotide sequence shown in SEQ ID NO.1.

[0026] Thirdly, the present invention seeks protection for the VdR4 gene having the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the full-length cDNA ORF of the gene in the sea island cotton material H7124 is shown in SEQ ID NO.1.

[0027] Fourthly, the present invention seeks protection for a protein encoded by the aforementioned VdR4 gene, the protein having the amino acid sequence shown in SEQ ID NO. 2.

[0028] Fifthly, the present invention seeks protection for biological materials containing the above-mentioned VdR4 gene, wherein the biological material is a recombinant vector, expression cassette, transgenic cell line or recombinant bacteria.

[0029] Studies have found that overexpressing the VdR4 gene in cotton significantly improves the plant's disease resistance without affecting its normal growth and development. Using the VdR4 gene as a target gene, cotton materials with significantly improved disease resistance were obtained. Furthermore, through hybridization and backcrossing with mainstream production varieties, the target gene / locus was introduced into existing promoted varieties to improve their disease resistance.

[0030] The advantages of this invention are as follows:

[0031] For the first time, the molecular mechanism by which the β,1,3-glucanase VdR4 in cotton interacts with the Verticillium wilt toxin PeVD1 enhances cotton's resistance to Verticillium wilt was identified. The important role of VdR4 in cotton's resistance to Verticillium wilt was clarified.

[0032] We analyzed the transcriptome of roots from *Verticillium wilt* V991-induced *Cotton Island* H7124 plants and identified a gene, VdR4, significantly upregulated by *Verticillium wilt*, encoding β,1,3-glucanase. RT-qPCR showed that this gene was induced by *Verticillium wilt* in both *Cotton Island* and upland cotton. In vitro antibacterial assays demonstrated that VdR4 has an inhibitory effect on *Verticillium wilt*. Subcellular localization showed that VdR4 is located in the cell membrane. Further studies showed that VdR4 can be secreted into the apoplast after salicylic acid induction. The full-length ORF sequence and encoded amino acid sequence of VdR4 were obtained from *Cotton Island* H7124 plants using PCR. We then used biotechnology to investigate the role of this gene in plant disease resistance and its functional mechanism, demonstrating that overexpression of this gene in cotton significantly improved plant disease resistance. By using VdR4 as a bait protein to screen interacting proteins in a yeast expression library of Verticillium wilt pathogen V991 induced by roots of H7124 sea island cotton, further full-length gene cloning and interaction verification were conducted. Combined with transgenic verification and molecular mechanism analysis, it was determined that VdR4 interacts with the Verticillium wilt pathogen toxin protein PeVD1 in cotton and participates in cotton Verticillium wilt resistance. Previous studies have reported that PeVD1 is an important effector factor in Verticillium wilt, which can target the cell nucleus and cause cytotoxicity. Our study clarified that PeVD1 is located in the cell membrane and nucleus. When PeVD1 and VdR4 were transiently co-expressed in tobacco, PeVD1 and VdR4 exhibited apoplast localization, indicating that VdR4 in the apoplast prevents PeVD1 from targeting the cell nucleus. The VdR4 overexpression cotton we created significantly improved cotton resistance to Verticillium wilt, providing theoretical guidance and genetic resources for improving cotton Verticillium wilt resistance through molecular design breeding or genetic engineering. Attached Figure Description

[0033] Figure 1 VdR4 expression was significantly upregulated in both Sea Island cotton H7124 and Upland cotton TM-1 induced by Verticillium wilt.

[0034] A. RT-qPCR was used to verify the change in VdR4 expression level in roots of upland cotton TM-1 after induction by Verticillium wilt. B. RT-qPCR was used to verify the change in VdR4 expression level in roots of island cotton H7124 after induction by Verticillium wilt. Points and error bars represent mean ± standard deviation (n = 3). All statistical analyses were performed using t-tests (** P < 0.01).

[0035] Figure 2 VdR4 inhibits the growth of Verticillium wilt in vitro.

[0036] A. The prokaryotically expressed and purified TF-VdR4 protein showed antibacterial activity against Verticillium wilt. Water and TF protein served as negative controls. B. The VdR4 protein lacked β,1,3-glucanase activity; TF (GST) protein served as a negative control. The previously reported Glu18 protein, possessing β,1,3-glucanase activity, served as a positive control. Statistical analyses were performed using t-tests (**P < 0.01; no significant difference in normal ranges).

[0037] Figure 3 VdR4 is located in the cell membrane and can be secreted into the apoplast after salicylic acid induction.

[0038] A. VdR4:GFP and cell membrane marker AtPIP2A:RFP co-localize. B. After plasmolysis, VdR4:GFP and cell membrane marker AtPIP2A:RFP co-localize on the cell membrane. C. After exogenous application of salicylic acid and plasmolysis, VdR4:GFP is secreted into the apoplast. Red arrows indicate the apoplast region. Scale bar: 50 μm.

[0039] Figure 4 Overexpression of VdR4 enhances resistance to Verticillium wilt in cotton.

[0040] A. RT-qPCR validation of VdR4 expression in pure cotton roots. Error bars represent mean ± standard deviation. B. VdR4 overexpression enhances cotton disease resistance. Phenotypic analysis of wild-type (W0) and VdR4-overexpressing (OE) plants. Photos were taken 15 and 20 days after Verticillium wilt inoculation. C. Percentage of wilted leaves in cotton after Verticillium wilt inoculation. Three biological replicates, each containing 10 seedlings. D. Vascular bundle discoloration phenotype in W0 and VdR4-overexpressing plants after Verticillium wilt inoculation. E. qPCR quantitative analysis of root fungal biomass in W0 and VdR4-overexpressing plants 15 days after Verticillium wilt inoculation. Error bars represent mean ± standard deviation. F. Fungal resuscitation experiment. Stem segments from plants 15 days after inoculation were cut and placed on potato dextrose agar plates, incubated at 25°C. Photos were taken on day 4 after incubation. All statistical analyses were performed using t-tests (* P < 0.05; ** P < 0.01).

[0041] Figure 5 VdR4 interacts with PeVD1, a protein that is effective against Verticillium wilt.

[0042] A. The interaction between PeVD1 and VdR4 was verified by a luciferase complementation assay. Only co-expression of cLuc-VdR4+PeVD1-nLuc produced a signal; the negative controls cLuc+nLuc, cLuc+PeVD1-nLuc, and cLuc-VdR4+nLuc did not produce a signal. B. PeVD1 interacts with VdR4 in Y2H. In a non-selective medium (SD / -Trp / -Leu), and in a selective medium (SD / -Trp / -Leu / -Ade / -His / Xa-gal), the interacting proteins grew and turned blue. The interactions of BD-Lam+AD-T and BD-53+AD-T served as negative and positive controls, respectively. C. BiFC experiments demonstrated the interaction between VdTRP and VdR4 in plant cells. Co-expression of p2YN-VdR4+p2YC-PeVD1 in Nicotiana benthamiana cells produced a YFP signal. Scale bar: 50 μm. D. Co-IP experiments demonstrated the interaction between VdTRP / VdR3 and VdTRP / VdR4. TRP-HA was co-expressed in *Nicotiana benthamiana*, expressing VdR3-FLAG and VdR4-Flag, respectively. Extracted proteins were purified using FLAG magnetic beads and detected by Western blotting with HA and Flag antibodies, respectively. VdR3 is a homolog of VdR4.

[0043] Figure 6 PeVD1 is crucial for the virulence of Verticillium wilt.

[0044] A. Changes in PeVD1 expression levels in Verticillium wilt after root induction in cotton. Expression data were converted to FPKM to calculate PeVD1 expression levels. Points and error bars represent mean ± standard deviation (n = 3). B. The virulence of the Δpevd1 mutant of Verticillium wilt to cotton was significantly reduced. Photographs were taken 15 days after inoculation with Verticillium wilt. C. Percentage of wilted cotton leaves at different days after inoculation with wild-type and Δpevd1 mutant strains of Verticillium wilt. V991 and two PeVD1 knockout strains, Δvd1-1 and Δvd1-5, were used. Points and error bars represent mean ± standard deviation (n = 3). D. PeVD1 localization in the cell membrane and nucleus. Scale bar: 50 μm. All statistical analyses were performed using t-tests (* P < 0.05; ** P < 0.01).

[0045] Figure 7 VdR4 interacts with PeVD1 in the apoplast, preventing PeVD1 from targeting the cell nucleus.

[0046] A. VdR4:GFP and PeVD1:RFP co-localize. B. After plasmolysis, VdR4:GFP and PeVD1:RFP co-localize in the apoplast. C. Extract apoplast proteins from tobacco samples co-injected with PeVD1-Flag, GFP, VdR3-GFP, and VdR4-GFP. PeVD1-Flag protein can only be detected if VdR3-GFP or VdR4-GFP is present. β-Actin is used as an internal standard protein. VdR3 is a homolog of VdR4. Detailed Implementation

[0047] The following examples illustrate specific implementation methods to further describe the content of the present invention in detail. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies that can be implemented in the art based on the above-mentioned content of the present invention should be included in the content of the present invention.

[0048] Example 1

[0049] (a) VdR4 expression was significantly upregulated in both Sea Island cotton H7124 and Upland cotton TM-1 induced by Verticillium wilt.

[0050] We identified a gene, VdR4, that was significantly upregulated by Verticillium wilt in the transcriptome of Verticillium wilt-induced cotton H7124, suggesting its potential involvement in cotton resistance to Verticillium wilt. VdR4 exists only once in the Verticillium wilt genome, within subgroup A. We designed a specific quantitative primer, DLVdR4, using cotton histone His3 as an internal standard primer, and verified by RT-qPCR that VdR4 was significantly induced by Verticillium wilt in the roots of both upland cotton TM-1 and Verticillium wilt-induced cotton H7124. Figure 1 (A, B). These results indicate that VdR4 plays an important role in the response of both upland and island cotton to Verticillium wilt. The primers used in the study are listed in Table 1.

[0051] Table 1: Primers used for amplification

[0052] Primer name Positive (5'-3') Reverse (5'-3') DLVdR4 AGGCATTGGGATATTGCTTCTAAG AAAAGACAAAACTCCCCAAATTCC His3 CGGTGGTGTGAAGAAGCCTCAT AATTTCACGAACAAGCCTCTGGAA

[0053] (ii) VdR4 has an in vitro antibacterial effect and can be secreted into the apoplast when induced by salicylic acid.

[0054] Domain analysis revealed that VdR4 encodes β,1,3-glucanase. β,1,3-glucanase is considered a class of disease-associated proteins and may have direct antibacterial effects. Using sea island cotton H7124 cDNA as a template and Sac I and Xba I as restriction enzyme sites, we constructed the prokaryotic expression vector pColdTF-VdR4 using TFVdR4 recombinant primers. pColdTF is a publicly available vector. TF-VdR4 protein was induced and purified through prokaryotic expression and showed antibacterial activity against Verticillium wilt spores (10... 5 In vitro antibacterial tests revealed that VdR4 had a significant antibacterial effect. Figure 2 A). However, interestingly, although VdR4 encodes β,1,3-glucanase, it lacks β,1,3-glucanase activity and cannot break down callosine ( Figure 2 B), indicating that VdR4 may inhibit Verticillium wilt by specifically recognizing the cell wall of the wilt disease. Using island cotton H7124 cDNA as a template and Kpn I and BamH I as restriction enzyme sites, we constructed the subcellular localization vector pBINGFP4-VdR4 using the pBINVdR4 recombinant primers. pBINGFP4 is a publicly available vector. Subcellular localization showed that VdR4 is localized to the cell membrane (B). Figure 3 A, B). After salicylic acid induction, VdR4 can be secreted into the apoplast ( Figure 3 C). Primers used for prokaryotic expression and subcellular localization were all vectors from publicly available literature. The primers used in the study are listed in Table 2.

[0055] Table 2: Primers used for amplification

[0056] Primer name Positive (5'-3') Reverse (5'-3') TFVdR4 recombinant primers CGATCGGGGAAATTCGAGCTCATGATTGGTGTTTGCTATGGGAT GAGAACACGGGGGACTCTAGATTACATATCAGATTGAAGAGGAAAGGTT pBINVdR4 recombinant primers ATTTACGAACGATAGGGTACCATGGCTATCTTTTCTTCATC GCCCTTGCTCACCATGGATCCGGACATATCAGATTGAAGAG

[0057] (III) VdR4 overexpression enhances cotton's disease resistance

[0058] We investigated whether VdR4 overexpression could enhance cotton resistance to Verticillium wilt. Driven by the 35S promoter, we obtained several transgenic cotton lines stably overexpressing VdR4 (using Sea Island cotton H7124 cDNA as a template, BamHI and SacHI as restriction sites, and constructing an overexpression vector using the pBI121-VdR4 recombinant primers), with pBI121 being a publicly available vector. OE66 and OE949, with the highest VdR4 expression levels, were selected for disease resistance identification. Figure 4 A). After inoculation with Verticillium wilt, compared with the control, the wilting rate of leaves in OE66 and OE949 was significantly reduced, the accumulation of pathogenic fungi in vascular tissue was less, and the root fungal biomass was lower. Figure 4 BE). Fungal recovery tests showed that, compared with the control, the stem fungal recovery of strains OE66 and OE949 infected with Verticillium wilt was less ( Figure 4F). The primers used in the study are listed in Table 3.

[0059] Table 3: Primers used for amplification

[0060] Primer name Positive (5'-3') Reverse (5'-3') pBI121-VdR4 recombinant primers ACGGGGGACTCTAGAGGATCCATGGCTATCTTTTCTTCATCCATGG CGATCGGGGAAATTCGAGCTCTTACATATCAGATTGAAGAGGAAAGGTT

[0061] (iv) VdR4 overexpression enhances cotton's disease resistance

[0062] To determine the molecular mechanism by which VdR4 participates in Verticillium wilt resistance, we used yeast two-hybrid (Y2H) technology to screen for proteins interacting with VdR4 from a cDNA library of Verticillium wilt induced by cotton roots (using Sea Island cotton H7124 cDNA as a template, constructing a vector with BDVdR4 recombinant primers, and EcoRI and BamHI restriction sites; this vector is a publicly reported vector). This experiment identified the interaction between the previously reported effector protein PeVD1 and VdR4. To verify the interaction between the two in plant cells, we performed a luciferase (Luc) complementation experiment in *Nicotiana benthamiana* (using V991 cDNA as a template, a vector was constructed using the pCambia1300-PeVD1-nLuc recombinant primers with BamHI and SaI I restriction sites; using *Cotton Island H7124* cDNA as a template, a vector was constructed using the pCambia1300-cLuc-VdR4 recombinant primers with KpnHI and BamHI restriction sites; pCambia1300-nLuc and pCambia1300-cLuc were publicly reported vectors). The luciferase (Luc) complementation experiment showed that luciferase signals were clearly detectable only at the infiltration sites co-expressing nLuc-PeVD1 / cLuc-VdR4, while no luciferase signal was detected in the negative control. Figure 5 A). Our Y2H study revealed that PeVD1 and VdR4 both interact ( Figure 5 B). We performed bimolecular fluorescence complementation (BIFC) experiments in *Nicotiana benthamiana* (using *Cotton Island H7124* cDNA as a template, constructing a vector with p2YN-VdR4 recombinant primers, with Pac I and Spe I restriction sites; using V991 cDNA as a template, constructing a vector with p2YC-PeVD1 recombinant primers, with Pac I and Spe I restriction sites; p2YN and p2YC are publicly reported vectors). The results showed that PeVD1 and VdR4 interact around the cell membrane. Figure 5 C). Co-IP experiments (using Sea Island cotton H7124 cDNA as a template, constructing a vector using pCambia1390-PeVD1-Flag recombinant primers, with Pst I and BamH I restriction sites; this vector is a publicly reported vector) also confirmed their interaction ( Figure 5 D). The primers used in the study are listed in Table 4.

[0063] Table 4: Primers used for amplification

[0064] Primer name Positive (5'-3') Reverse (5'-3') pCambia1300-PeVD1-nLuc recombinant primers CTCGGTACCCGGGGATCCATGCAGTTCACCCTCGCCG GTACGAGATCTGGTCGACAGCCTCGGCGGGAGCGTC pCambia1300-cLuc-VdR4 recombinant primers GCGTCCCGGGGCGGTACCATGGCTATCTTTTCTTCATCCATGG AGTCCATTTGTTGGATCCTTACATATCAGATTGAAGAGGAAAGGTT BDVdR4 recombinant primers ATGGCCATGGAGGCCGAATTCATGATTGGTGTTTGCTATGGGAT CCGCTGCAGGTCGACGGATCCCATATCAGATTGAAGAGGAAAGGTTG ADPeVD1 recombinant primers GCCATGGAGGCCAGTGAATTCATGCAGTTCACCCTCGCCG CAGCTCGAGCTCGATGGATCCAGCCTGGGCGGGAGCGTC p2YN-VdR4 recombinant primers ATTTACGAACGATAGTTAATTAAATGGCTATCTTTTCTTCATCCATGG ACTGCCACCTCCTCCACTAGTCATATCAGATTGAAGAGGAAAGGTTG p2YC - PeVD1 recombinant primer ATTTACGAACGATAGTTAATTAAATGCAGTTCACCCTCGCCG ACTGCCACCTCCTCCACTAGTAGCCTCGGCGGGAGCGTC pCambia1390 - PeVD1 - Flag TTCTGCACTAGGTACCTGCAGATGCAGTTCACCCTCGCCG GATGGATCCGTCGACCTGCAGAGCCTGGGCGGGAGCGTC

[0065] (v) PeVD1 is crucial to the toxicity of Verticillium wilt. VdR4 interacts with PeVD1 in the apoplast, preventing it from targeting the cell nucleus.

[0066] Transcriptome analysis of root-induced Verticillium wilt V991 showed that PeVD1 was significantly induced ( Figure 6 A). Using V991 DNA as a template, we used VD1shang (Kpn I and BamH I) and VD1xia (Xba I and Hind III) primers (pDHT2 is a publicly reported vector) to knock out PeVD1 in Verticillium wilt using homologous recombination. The mutant showed a significant reduction in its ability to infect cotton. Figure 6 (B, C). Using V991 cDNA as a template, we constructed PeVD1 subcellular localization vectors (Xba Ⅰ and BamH Ⅰ) using pBIN-VD1 primers. Subcellular localization results showed that PeVD1 was localized in the cell membrane and nucleus, which is consistent with previous reports.

[0067] Using V991 cDNA as a template, we constructed vectors (Xba I and SaI I) carrying the red fluorescent tag RFP for PeVD1 using VD1RFP recombinant primers. pCAMBIA1300-RFP was a publicly reported vector. Subcellular localization results showed that PeVD1 co-localized with VdR4 (…). Figure 7 A). Plasmolysis results indicate that PeVD1 and VdR4 are co-localized in the apoplast ( Figure 7 B). We constructed the PeVD1-Flag vector (Xba Ⅰ) using V991 cDNA as a template and the PeVD1-Flag recombinant primers, with pCAMBIA1300-Flag as a publicly reported vector. We extracted apoplast proteins from PeVD1-Flag co-expressed with GFP, VdR4 homologs VdR3-GFP, and VdR4-GFP, respectively. The results showed that the apoplast proteins contained PeVD1 only when PeVD1 was co-expressed with VdR3-GFP or VdR4-GFP; while when PeVD1 was co-expressed with empty GFP, PeVD1 was undetectable in the apoplast proteins. Figure 7 C), the amino acid sequence of the protein encoded by VdR3, a homolog of VdR4, is shown in SEQ ID NO.3.

[0068] These results indicate that VdR4 interacts with PeVD1 in the aplast, preventing it from targeting the cell nucleus and mitigating its toxicity to plant cells. The primers used in this study are listed in Table 5.

[0069] Table 5: Primers used for amplification

[0070] Primer name Forward (5’ - 3’) Reverse (5’ - 3’) VD1shang TACGAATTCGAGCTCGGTACCCCTCTTTGGGCCAACTCTCG GCTCCTTCAATATCAGGATCCTTTGATGTTGGGTGAGGTTGAA VD1xia GCCGACCGGGAACCATCTAGAGCGTTGCATTTCGGATAGAAA GCCAAGCTTGCATGCCTGCAGTAGTTAGCGGCGATCCTGTTG pBIN - VD1 recombinant primer AAGTCCGGAGCTAGCTCTAGAATGCAGTTCACCCTCGCCG GCCCTTGCTCACCATGGATCCAGCCTCGGCGGGAGCGTC VD1RFP recombinant primer GGTACCCGGGGATCCTCTAGAATGCAGTTCACCCTCGCCG CTCGGAGGAGGCCATGTCGACAGCCTGGGCGGGAGCGTC PeVD1 - Flag recombinant primer TTCTGCACTAGGTACCTGCAGATGCAGTTCACCCTCGCCG GATGGATCCGTCGACCTGCAGAGCCTGGGCGGGAGCGTC

[0071] cDNA ORF sequence of VdR4 in Sea Island cotton H7124

[0072]

[0073] Amino acid sequence of VdR4 in Gossypium barbadense H7124

[0074] MAIFSSSMAAMLLLGLLTANLDPTVAQIGVCYGMLGNNLPNAREVINLYKSNNIKRMRLYDPNQQALQALRGSNIEVILGVPNDQLQNLANPSKAKSWVRSNVVAYWPRVRFRYIAVGNEVPPSSSLAQFVLPALVNVFNAVRSAGLESQIKVSIAIDMTLIGVSYPPSAGAFRGDVRSYLDPIIGHLAWARTALLANIYTYFSYSGNPRDISLPYALFTSPSPIVWDQGRGYQNLFDAMLDSLYSALEKAGQCGLEVVVSESGWPSAGGFGTSVDNAATYLSNLIKHVQKGTPKRPGKAIETYLFALFDENQKSGPELERHFGLFSPNKQPKYQLHFGGGRHWHIASKEYNATFPLQSDM

[0075] Protein sequence of VdR3 in Gossypium barbadense H7124 (GB_A09G0943)

[0076] MQTMAIFSTSMAAMLLLLGLFAANLDPTAAQIGVCYGMLGNNLPNSWEVIQLYKSNNIRRLRLYDPNQQALQALRGSNIEVMLGVPNDQLQNLADPSKARSWVQSNVVAYWPSVRFRYIAVGNEVPHSSWLAQFVLPALVNVFNAVRSAGLESQIKVSIAIDMTLIGVSYPPSAGAFRGDVRSYLDPIIGHLAWARTPLLANIYTYFSYSGNPRDISLPYALFTSPSPVVWDQGRGYQNLFDAMLDSLYSALEKAGQGGLEVVVSESGWPSAGGFGTSIDNAATYLSNLIRHVQGGTPKRPGKAIETYLFALFDENSKPGPELERHFGLFSPNKQPKYQLHFGGGRHWDIASEEYNGTFPLKSDM。

Claims

1. The overexpressed nucleotide sequence is shown in SEQ ID NO.

1. VdR4 The application of genes in the following a1) or a2) : al) Improve cotton resistance to Verticillium wilt; a2) Develop new cotton germplasm with improved resistance to Verticillium wilt.

2. Promotes the use of nucleotide sequences as shown in SEQ ID NO.1 VdR4 Applications of gene expression biological materials in the following a1) or a2) processes: al) Improve cotton resistance to Verticillium wilt; a2) Develop new cotton germplasm with improved resistance to Verticillium wilt; The biomaterial is any one of the following (b1) to (b6): b1) contains the above VdR4 Gene expression cassettes; b2) contains the above VdR4 Recombinant vectors containing genes, or recombinant vectors containing the expression cassette described in b1); b3) contains the above VdR4 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in b1), or recombinant microorganisms containing the recombinant vector described in b2); b4) contains the above VdR4 A transgenic plant cell line containing the gene, or a transgenic plant cell line containing the expression cassette described in b1), or a transgenic plant cell line containing the recombinant vector described in b2); b5) contains the above VdR4 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in b1), or transgenic plant tissue containing the recombinant vector described in b2); b6) contains the above VdR4 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in b1), or transgenic plant organs containing the recombinant vector described in b2).

3. The application according to claim 1 or 2, characterized in that, As stated VdR4 Genes are used as target genes, and through genetic engineering methods, they are overexpressed. VdR4 Genes to improve cotton resistance to Verticillium wilt or to cultivate new cotton germplasm with improved resistance to Verticillium wilt.

4. A method for improving cotton resistance to Verticillium wilt, characterized in that: Overexpression of nucleotide sequences as shown in SEQ ID NO.1 in cotton. VdR4 Gene.

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