Application of VdR4 gene in improving disease resistance of cotton verticillium wilt

Abstract

The invention discloses application of a VdR4 gene in improving the disease resistance of cotton verticillium wilt, and belongs to the field of biotechnology application. The VdR4 gene provided by the invention is used for coding beta, 1, 3-glucanase. The invention provides a full-length ORF (Open Reading Frame) nucleotide sequence and an amino acid sequence of VdR4 in an allotetraploid sea island cotton material H7124. The cotton VdR4 interacts with the verticillium wilt effect protein PeVD1 and participates in cotton verticillium wilt resistance. The VdR4 is induced by verticillium dahliae in cotton roots to realize obvious up-regulation expression. Overexpression of the gene in cotton can significantly improve plant disease resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology application, and relates to the application of cotton VdR4 gene in improving the disease resistance of cotton to Verticillium wilt. By analyzing the transcriptome of the root of Sea Island Cotton H7124 induced by Verticillium wilt fungus V991, a gene VdR4 whose expression is significantly upregulated by Verticillium wilt fungus is identified, and the gene encodes β,1,3-glucanase. RT-qPCR analysis shows that the expression of the gene in both Sea Island Cotton and Upland Cotton materials is induced by Verticillium wilt fungus. In vitro antibacterial test shows that VdR4 has an in vitro antibacterial effect on Verticillium wilt fungus. Subcellular localization shows that VdR4 is located in the cell membrane. Further research shows that after salicylic acid induction, VdR4 can be secreted into the apoplast. The full-length ORF sequence and the encoded amino acid sequence of VdR4 are obtained in Sea Island Cotton H7124 by using PCR technology. The plant disease resistance and functional mechanism involved in the gene are specifically studied by using biotechnology, and the overexpression of the gene in cotton significantly improves the disease resistance of the plant. By using VdR4 as bait protein to screen for interacting proteins in the yeast expression library of Verticillium wilt V991 constructed by root induction of cotton H7124, the full-length gene sequence was cloned and the interaction was verified. Combined with transgenic verification and molecular mechanism analysis, it was confirmed that VdR4 in cotton interacts with the Verticillium wilt toxic protein PeVD1 and participates in cotton Verticillium wilt resistance. Studies have reported that PeVD1 is an important effector factor in Verticillium wilt, which can target the nucleus and cause toxicity to cells. Our study confirmed that PeVD1 is localized in the cell membrane and nucleus. When PeVD1 and VdR4 are co-expressed in tobacco, PeVD1 and VdR4 are localized in the apoplast, indicating that VdR4 in the apoplast prevents PeVD1 from targeting the nucleus. Overexpression of VdR4 in cotton significantly improves cotton resistance to Verticillium wilt, and a new cotton germplasm with high resistance to Verticillium wilt is obtained. Background Art

[0002] Verticillium wilt of cotton is an important disease in cotton production and one of the objects of national agricultural plant quarantine. In 1914, the United States first reported that the disease caused a yield loss of 0.5-3.5% in the United States. At present, the disease has spread to major cotton producing areas around the world. Since my country introduced cotton from the United States in the 1930s, Verticillium wilt has also been introduced to China. In the early days, Verticillium wilt only caused harm in some provinces, but after 1990, the disease has spread to all cotton-growing areas in the country. It is estimated that the area of ​​Verticillium wilt in China is about 2.667 million hectares per year, accounting for about half of the country's cotton planting area, 1.333 million hectares of seriously diseased fields, and about 2 million dan of lint are lost each year. Therefore, Verticillium wilt of cotton has become the main obstacle to the sustained, stable and high yield of cotton in my country. Verticillium wilt of cotton is a serious vascular disease caused by the soil-borne fungus Verticillium dahliae. After being infected by Verticillium wilt, the leaves of cotton lose their green color and turn yellow until they wilt and fall off, and even cause the death of the plant, which seriously reduces the yield and fiber quality of cotton. For Verticillium wilt disease, chemical control strategies are not ideal, and management methods such as field rotation can only alleviate the occurrence of the disease appropriately. Discovering key disease-resistant genes in cotton and creating disease-resistant materials are the most direct and effective strategies to improve cotton's resistance to Verticillium wilt.

[0003] Identification of resistance sources has successfully improved resistance to Verticillium wilt in other crop varieties. 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, Sea Island cotton is much more resistant to infection with Verticillium dahliae than the most widely planted upland cotton variety in the world. However, compared with upland cotton, Sea Island cotton has a lower yield, a smaller planting area, and lower economic benefits. Therefore, researchers can discover key genes for resistance to Verticillium wilt from Sea Island cotton and cultivate excellent transgenic upland cotton varieties resistant to Verticillium wilt through molecular breeding. Zhang et al. (2007) cloned a tomato VE homologous gene, GbVE1, from a Sea Island cotton variety resistant to Verticillium wilt. The study found that the GbVE1 gene was 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 induction rate of GbVE1 in the disease-resistant Sea Island cotton was faster and the induction effect was much stronger than that in the susceptible upland cotton. The GbVE1 gene was transformed into Arabidopsis thaliana and upland cotton through Agrobacterium-mediated method, and the transgenic Arabidopsis thaliana and upland cotton had increased resistance to Verticillium wilt.

[0004] The life cycle of Verticillium wilt begins with the germination of microsclerotia. When the secretions released by the plant roots are sensed as a signal, the microsclerotia begin to produce germ tubes and extend longitudinally along the root epidermal cells. A small number of hyphae swell at the top and adhere tightly to the root surface in the form of attachment branches, forming a narrow infection nail structure that penetrates the epidermis for infection. During the infection process, the pathogen will secrete some colloids to firmly fix its attachment branches on the infection interface to withstand the recoil generated at the moment of penetration. At the same time, a large number of cell wall hydrolases, such as pectinases, cutinases and cellulases, are released to destroy plant cell wall tissues. A large number of effector factors destroy or interfere with the plant's immune response. After successful invasion, the hyphae gradually spread to the center, eventually entering the xylem and colonizing, growing and multiplying in or between cells, and extending upward along the xylem vessels to the stems or leaves.

[0005] In addition, certain effector proteins secreted by Verticillium dahliae are also important "accomplices" in the infection of hosts by Verticillium dahliae. Similar to other fungal pathogens, the genome of Verticillium dahliae encodes more than 700 secreted proteins. Wang et al. (2020) systematically studied the transient expression of predictable fungal effector proteins (including 123 scp) in the V991 strain, discovered three effectors that cause cell death, and demonstrated that they require BAK1 and SOBIR1 to trigger host immunity. Kombrink et al. (2017) studied LysM effectors in Verticillium dahliae. Through comparative genomics, they identified three core LysM effectors that are preserved in a collection of Verticillium dahliae strains. However, they demonstrated that these three effectors did not contribute to virulence in host plants of different classifications. Interestingly, they found a lineage-specific LysM effector in Verticillium dahliae VdLs17 that is virulent to tomatoes but not to Arabidopsis. Zhang et al. (2017) discovered an effector protein (VdSCP7) in the Verticillium dahliae strain V592 that can target the plant cell nucleus and regulate immunity to fungal infection.

[0006] In summary, the genes related to cotton Verticillium wilt resistance were screened out, which provided 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 the present invention is to provide an application of cotton VdR4 gene in improving cotton disease resistance or cultivating new cotton germplasm with improved disease resistance. Cotton materials with VdR4 gene transfection have been used to prove that overexpression of VdR4 can significantly improve plant resistance to Verticillium wilt. Using this gene as a target gene, through genetic engineering methods such as transgenic, the VdR4 gene is overexpressed in plants, and new cotton germplasm with normal growth and development, no significant difference in cotton fiber yield and quality compared with the control, and significantly improved resistance is cultivated and applied in production.

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

[0009] Another object of the present 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 in the sea island cotton material H7124 and the amino acid sequence of the encoded protein.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] In the first aspect, the present invention claims the use of the VdR4 gene or 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 disease resistance;

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

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

[0015] Furthermore, in the above application, the substance that promotes the expression of the VdR4 gene or the substance that 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) a recombinant microorganism containing the VdR4 gene, or a recombinant microorganism containing the expression cassette described in b1), or a recombinant microorganism 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 application, the protein encoded by the VdR4 gene is a protein having an amino acid sequence as shown in SEQ ID NO.2.

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

[0024] Furthermore, the disease resistance is Verticillium wilt resistance.

[0025] In a second aspect, the present invention claims a method for improving cotton's resistance to Verticillium wilt, which comprises overexpressing the VdR4 gene in cotton; the VdR4 gene is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.1.

[0026] In the third aspect, the present invention seeks to protect the VdR4 gene, which has a nucleotide sequence as shown in SEQ ID NO.1; the gene is in the sea island cotton material H7124, and the nucleotide sequence of its full-length cDNA ORF is shown in SEQ ID NO.1.

[0027] In a fourth aspect, the present invention claims protection for a protein encoded by the above-mentioned VdR4 gene, which has an amino acid sequence as shown in SEQ ID NO.2.

[0028] In a fifth aspect, the present invention seeks to protect a biological material containing the above-mentioned VdR4 gene, wherein the biological material is a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

[0029] The study found that overexpressing the VdR4 gene in cotton can significantly improve the disease resistance of the plant without affecting the normal growth and development of the plant. The VdR4 gene is used as a target gene to obtain cotton materials with significantly improved disease resistance, and through hybridization and backcrossing with the main promoted varieties in production, the target gene / site is introduced into the existing promoted varieties to improve the disease resistance of the promoted varieties.

[0030] The advantages of the present invention are:

[0031] For the first time, the molecular mechanism of the interaction between cotton β,1,3-glucanase VdR4 and Verticillium wilt toxic protein PeVD1 was identified to improve cotton resistance to Verticillium wilt, and the important role of VdR4 in cotton resistance to Verticillium wilt was clarified.

[0032] We analyzed the transcriptome of the roots of Cotton (Gossypium hirsutum H7124) induced by Verticillium dahliae V991 and identified a gene VdR4 that was significantly upregulated by Verticillium dahliae. This gene encodes β,1,3-glucanase. RT-qPCR showed that the gene was induced by Verticillium dahliae in both Cotton (Gossypium hirsutum) and Upland cotton. In vitro antibacterial tests showed that VdR4 had an in vitro antibacterial effect on Verticillium wilt. Subcellular localization showed that VdR4 was localized in the cell membrane. Further studies showed that after salicylic acid induction, VdR4 could be secreted into the apoplast. The full-length ORF sequence and the encoded amino acid sequence of VdR4 were obtained in Cotton (Gossypium hirsutum H7124) by PCR. The plant disease resistance and functional mechanism involved in this gene were specifically studied by biotechnology, and overexpression of this gene in cotton significantly improved plant disease resistance. By using VdR4 as bait protein to screen for interacting proteins in the yeast expression library of Verticillium wilt V991 constructed by root induction of sea island cotton material H7124, the full-length gene sequence was cloned and the interaction was verified. Combined with transgenic verification and molecular mechanism analysis, it was confirmed that VdR4 in cotton interacts with the Verticillium wilt toxic protein PeVD1 and participates in cotton Verticillium wilt resistance. Studies have reported that PeVD1 is an important effector factor in Verticillium wilt and can target the cell nucleus to cause toxicity to cells. Our study confirmed that PeVD1 is located in the cell membrane and nucleus. When PeVD1 and VdR4 are co-transiently expressed in tobacco, PeVD1 and VdR4 appear in the apoplast, indicating that VdR4 in the apoplast prevents PeVD1 from targeting the cell nucleus. The VdR4 overexpression cotton we created significantly improved cotton's resistance to Verticillium wilt, providing theoretical guidance and genetic resources for improving cotton Verticillium wilt resistance through molecular design breeding or genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 VdR4 was significantly upregulated in both sea island cotton H7124 and upland cotton TM-1 induced by Verticillium dahliae.

[0034] A, RT-qPCR validation of the expression level of VdR4 in the roots of upland cotton TM-1 after induction by Verticillium dahliae. B, RT-qPCR validation of the expression level of VdR4 in the roots of sea island cotton H7124 after induction by Verticillium dahliae. 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 dahliae in vitro.

[0036] A. The prokaryotic expressed and purified TF-VdR4 protein has an antibacterial effect on Verticillium dahliae. Water and TF protein were used as negative controls. B. VdR4 protein has no β,1,3-glucanase activity, TF (GST) protein was used as a negative control, and Glu18 protein, which has been reported to have β,1,3-glucanase activity, was used as a positive control. Statistical analysis was performed using t-test (**P<0.01; NS not significant).

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

[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 salicylic acid is applied, VdR4:GFP is secreted into the apoplast after plasmolysis. The red arrow indicates the apoplast region. Scale bar: 50μm.

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

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

[0041] Figure 5 VdR4 interacts with the Verticillium wilt effector protein PeVD1.

[0042] A. Luciferase complementation assay to verify PeVD1 / VdR4 interaction. Only cLuc-VdR4+PeVD1-nLuc co-expression produced a signal, while negative controls cLuc+nLuc, cLuc+PeVD1-nLuc, and cLuc-VdR4+nLuc did not produce a signal. B. PeVD1 interacts with VdR4 in Y2H. Two deficiencies (SD / -Trp / -Leu) were used as non-selective medium, and four deficiencies (SD / -Trp / -Leu / -Ade / -His / Xa-gal) were used as selective medium. The interacting proteins could grow and turn blue. The interactions of BD-Lam+AD-T and BD-53+AD-T were used as negative and positive controls, respectively. C. BiFC assay showed the interaction between VdTRP and VdR4 in plant cells. Co-expression of p2YN-VdR4+p2YC-PeVD1 in Nicotiana benthamiana cells produced YFP signals. Scale bar: 50 μm. D. Co-IP experiments demonstrated that VdTRP / VdR3 and VdTRP / VdR4 interact with each other. TRP-HA, VdR3-FLAG and VdR4-Flag were co-expressed in Nicotiana benthamiana. The extracted proteins were purified with FLAG magnetic beads and detected by Western blotting with HA antibody and Flag antibody. VdR3 is a homologous gene of VdR4.

[0043] Figure 6 PeVD1 is essential for the virulence of Verticillium dahliae.

[0044] A, Changes in the expression level of PeVD1 in Verticillium dahliae after induction in cotton roots. The expression data were converted to FPKM to calculate the expression of PeVD1. Points and error bars represent mean ± standard deviation (n = 3). B, The virulence of the Δpevd1 mutant of Verticillium dahliae to cotton was significantly reduced. Photos were taken 15 days after inoculation with Verticillium dahliae. C, The percentage of wilting leaves in cotton on different days after inoculation with the wild-type and Δpevd1 mutant strains of Verticillium dahliae. 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 is localized in the cell membrane and nucleus. Scale bar: 50 μm. All statistical analyses were performed using t-test (*P < 0.05; **P < 0.01).

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

[0046] A. VdR4:GFP and PeVD1:RFP colocalize. B. VdR4:GFP and PeVD1:RFP colocalize in the apoplast after plasmolysis. C. Extraction of tobacco apoplast proteins co-injected with PeVD1-Flag, GFP, VdR3-GFP, and VdR4-GFP. PeVD1-Flag protein can only be detected in the presence of VdR3-GFP or VdR4-GFP. β-Actin is used as an internal standard protein. VdR3 is a homologous gene of VdR4. DETAILED DESCRIPTION

[0047] The following is a detailed description of the present invention by presenting specific implementation methods in the form of examples. 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 belong to the content of the present invention.

[0048] Example 1

[0049] (A) VdR4 was significantly upregulated in both sea island cotton H7124 and upland cotton TM-1 induced by Verticillium dahliae.

[0050] We identified a gene VdR4 that was significantly induced and upregulated by Verticillium wilt from the transcriptome of Sea Island Cotton H7124 induced by Verticillium wilt, indicating that it may be involved in cotton resistance to Verticillium wilt. VdR4 is only one in the Sea Island Cotton genome and exists in subgroup A. We designed a specific quantitative primer DLVdR4 and used cotton histone His3 as the internal standard primer. RT-qPCR verified that VdR4 was significantly induced by Verticillium wilt in the roots of both Upland Cotton TM-1 and Sea Island Cotton H7124 ( Figure 1 A, B). These results indicate that VdR4 plays an important role in the response of both upland cotton and sea island cotton to Verticillium wilt. The primers used in the study are listed in Table 1.

[0051] Table 1: Primers used for amplification

[0052]

[0053] (ii) VdR4 has an antibacterial effect in vitro and can be secreted into the apoplast under the induction of salicylic acid.

[0054] Domain analysis showed that VdR4 encodes β,1,3-glucanase. β,1,3-glucanase is considered to be a type of pathogenesis-related protein and may have a direct antibacterial effect. We used the cDNA of sea island cotton H7124 as a template, SacⅠ and XbaⅠ as restriction sites, and used TFVdR4 recombinant primers to construct a prokaryotic expression vector pColdTF-VdR4. pColdTF is a public vector. TF-VdR4 protein was induced and purified by prokaryotic expression, and the expression of TF-VdR4 protein was inhibited against Verticillium wilt spores (10 5 ) were used for in vitro antibacterial testing and found that VdR4 had a significant antibacterial effect ( Figure 2 A). However, it is interesting that although VdR4 encodes β,1,3-glucanase, it has no β,1,3-glucanase activity and cannot degrade callose ( Figure 2 B), which indicates that VdR4 may inhibit Verticillium wilt by specifically recognizing the cell wall. We used the cDNA of sea island cotton H7124 as a template, KpnⅠ and BamHⅠ as restriction sites, and used pBINVdR4 recombination primers to construct the subcellular localization vector pBINGFP4-VdR4 of VdR4. pBINGFP4 is a public vector. Subcellular localization showed that VdR4 was located in the cell membrane ( Figure 3 A, B). After salicylic acid induction, VdR4 can be secreted into the apoplast ( Figure 3 C). The primers used for prokaryotic expression and subcellular localization were all vectors from public literature. The primers used in the study are listed in Table 2.

[0055] Table 2: Primers used for amplification

[0056]

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

[0058] We studied whether VdR4 overexpression can improve cotton resistance to Verticillium wilt. Driven by the 35S promoter, we obtained several transgenic cotton lines that stably overexpressed VdR4 (using the cDNA of sea island cotton H7124 as a template, BamHI and SacⅠ as restriction sites, and using pBI121-VdR4 recombination primers to construct an overexpression transgenic vector), pBI121 is a public 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 leaf wilting rate of OE66 and OE949 was significantly reduced, the accumulation of pathogenic fungi in the vascular tube was less, and the fungal biomass in the root system was less ( Figure 4 BE). Fungal recovery experiments showed that the fungal recovery in the stems of lines OE66 and OE949 infected with Verticillium dahliae was less than that in the control ( Figure 4 F). The primers used in the study are listed in Table 3.

[0059] Table 3: Primers used for amplification

[0060]

[0061] (IV) VdR4 overexpression enhances cotton disease resistance

[0062] In order to determine the molecular mechanism of VdR4's involvement in Verticillium wilt resistance, we used yeast two-hybrid (Y2H) technology to screen for VdR4-interacting proteins from the cDNA library of cotton root-induced Verticillium dahliae (G. barbadense H7124 cDNA was used as a template, and the vector was constructed using BDVdR4 recombinant primers, with restriction sites of EcoRⅠ and BamHⅠ, and the vector was a publicly reported vector). This experiment identified the interaction between the reported effector protein PeVD1 of Verticillium dahliae and VdR4. In order to verify the interaction between the two in plant cells, we conducted a luciferase (Luc) complementation experiment in Nicotiana benthamiana (using V991 cDNA as a template, using pCambia1300-PeVD1-nLuc recombination primers to construct a vector, with restriction sites of BamHⅠ and SaIⅠ; using sea island cotton H7124 cDNA as a template, using pCambia1300-cLuc-VdR4 recombination primers to construct a vector, with restriction sites of KpnⅠ and BamHⅠ; pCambia1300-nLuc and pCambia1300-cLuc are publicly reported vectors). The luciferase (Luc) complementation experiment showed that the luciferase signal could only be clearly monitored in the infiltration site co-expressing nLuc-PeVD1 / cLuc-VdR4, while no luciferase signal was detected in the negative control ( Figure 5 A). Through Y2H studies, we found that PeVD1 interacts with VdR4 ( Figure 5 B). We conducted a bimolecular fluorescence complementation (BIFC) experiment in Nicotiana benthamiana (using the cDNA of Gossypium barbadensis H7124 as a template, using the p2YN-VdR4 recombinant primer to construct a vector, with restriction sites PacⅠ and SpeⅠ; using the V991 cDNA as a template, using the p2YC-PeVD1 recombinant primer to construct a vector, with restriction sites PacⅠ and SpeⅠ; p2YN and p2YC are publicly reported vectors). The results showed that PeVD1 interacted with VdR4 around the cell membrane ( Figure 5 C). Co-IP experiments (using sea island cotton H7124 cDNA as a template, using pCambia1390-PeVD1-Flag recombination primers to construct a vector, with restriction sites of PstⅠ and BamHI, and 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]

[0065]

[0066] (V) PeVD1 is essential for the toxicity of Verticillium wilt, and VdR4 interacts with PeVD1 in the apoplast, preventing it from targeting the nucleus.

[0067] Transcriptome analysis of root-inducing Verticillium dahliae V991 showed that PeVD1 was significantly induced ( Figure 6 A). We used V991 DNA as a template, VD1shang (KpnⅠ and BamHⅠ) and VD1xia (XbaⅠ and HindⅢ) primers (pDHT2 is a public reporting vector), and used homologous recombination to knock out PeVD1 in Verticillium wilt. The mutant's ability to infect cotton was significantly reduced ( Figure 6 B, C). We used V991 cDNA as a template and pBIN-VD1 primers to construct a PeVD1 subcellular localization vector (XbaⅠ and BamHⅠ). The subcellular localization results showed that PeVD1 was localized in the cell membrane and nucleus, which was consistent with previous reports.

[0068] We used V991 cDNA as a template and VD1RFP recombination primers to construct a PeVD1 vector carrying the red fluorescent tag RFP (XbaⅠ and SaIⅠ), and pCAMBIA1300-RFP was used as a public reporting vector. Subcellular localization results showed that PeVD1 colocalized with VdR4 ( Figure 7 A). Plasmolysis results showed that PeVD1 and VdR4 co-localized in the apoplast ( Figure 7 B). We used V991 cDNA as a template and PeVD1-Flag recombination primers to construct the PeVD1-Flag vector (XbaⅠ), and pCAMBIA1300-Flag was used as a public reporting vector. We extracted the apoplast proteins of PeVD1-Flag co-expressed with GFP, VdR4 homologous genes VdR3-GFP and VdR4-GFP, respectively. The results showed that only when PeVD1 was co-expressed with VdR3-GFP or VdR4-GFP, the apoplast proteins contained PeVD1; when PeVD1 was co-expressed with empty GFP, PeVD1 could not be detected in the apoplast proteins ( Figure 7 C), the amino acid sequence of the protein encoded by VdR3, a homologous gene of VdR4, is shown in SEQ ID NO.3.

[0069] These results indicate that VdR4 interacts with PeVD1 in the apoplast, preventing it from targeting to the nucleus and alleviating its toxicity to plant cells. The primers used in the study are listed in Table 5.

[0070] Table 5: Primers used for amplification

[0071]

[0072]

[0073] cDNA ORF Sequence of VdR4 in Gossypium barbadense H7124

[0074]

[0093] Amino acid sequence of TAAVdR4 in Gossypium barbadense H7124

[0094] MAIFSSSMAAMLLLGLLTANLDPTVAQIGVCYGMLGNNLPNAREVINLYKSNNIKRMRLYDPNQQALQALRGSNIEVILGVPNDQLQNLANPSKAKSWVRSNVVAYWPRVRFRYIAVGNEVPPSSSLAQFVLPALVNVFNAVRSAGLESQIKVSIAIDMTLIGVSYPPSAGAFRGDVRSYLDPIIGHLAWARTALLANIYTYFSYSGNPRDISLPYALFTSPSPIVWDQGRGYQNLFDAMLDSLYSALEKAGQCGLEVVVSESGWPSAGGFGTSVDNAATYLSNLIKHVQKGTPKRPGKAIETYLFALFDENQKSGPELERHFGLFSPNKQPKYQLHFGGGRHWHIASKEYNAT

[0100] Protein sequence of FPLQSDMVdR3 in Gossypium barbadense H7124 (GB_A09G0943)

[0101] MQTMAIFSTSMAAMLLLLGLFAANLDPTAAQIGVCYGMLGNNLPNSWEVIQLYKSNNIRRLRLYDPNQQALQALRGSNIEVMLGVPNDQLQNLADPSKARSWVQSNVVAYWPSVRFRYIAVGNEVPHSSWLAQFVLPALVNVFNAVRSAGLESQIKVSIAIDMTLIGVSYPPSAGAFRGDVRSYLDPIIGHLAWARTPLLANIYTYFSYSGNPRDISLPYALFTSPSPVVWDQGRGYQNLFDAMLDSLYSALEKAGQGGLEVVVSESGWPSAGGFGTSIDNAATYLSNLIRHVQGGTPKRPGKAIETYLFALFDENSKPGPELERHFGLFSPNKQPKYQLHFGGGRHWDIASEEYNGTFPLKSDM。

Claims

1. Use of the VdR4 gene or 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): al) Improve cotton disease resistance; a2) Cultivate new cotton germplasm with improved disease resistance; The VdR4 gene is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The substance that promotes the expression of the VdR4 gene or the substance that 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): b1) an expression cassette containing the VdR4 gene; b2) a recombinant vector containing the VdR4 gene, or a recombinant vector containing the expression cassette described in b1); b3) a recombinant microorganism containing the VdR4 gene, or a recombinant microorganism containing the expression cassette described in b1), or a recombinant microorganism containing the recombinant vector described in b2); 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); 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); 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).

3. The use according to claim 1 or 2, characterized in that: The protein encoded by the VdR4 gene is a protein having an amino acid sequence as shown in SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The VdR4 gene is used as a target gene and overexpressed through genetic engineering methods to improve the disease resistance of cotton or cultivate new cotton germplasm with improved disease resistance.

5. The use according to claim 1 or 4, characterized in that: The disease resistance is Verticillium wilt resistance.

6. A method for improving cotton Verticillium wilt resistance, characterized in that: The VdR4 gene is overexpressed in cotton; the VdR4 gene is a DNA molecule having a nucleotide sequence as shown in SEQ ID NO.

1.

7. A VdR4 gene having a nucleotide sequence as shown in SEQ ID NO.

1.

8. The protein encoded by the VdR4 gene according to claim 7, which has the amino acid sequence shown in SEQ ID NO.

2.

9. The biological material containing the VdR4 gene according to claim 7, characterized in that: The biological material is a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium.

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