VdSP8, a large liriomyza exoprotein elicitor, and application thereof

Abstract

The application provides a Verticillium dahliae exoprotein elicitor VdSP8, and finds that VdSP8 can improve plant resistance and induce plant defense response, heterologous expression of VdSP8 significantly improves the resistance of host plants to multiple pathogenic bacteria, including Verticillium dahliae, Botrytis cinerea and Pseudomonas syringae; meanwhile, VdSP8 can also improve plant growth rate, increase fresh weight and promote plant bolting. The results of cytological studies show that VdSP8 improves the resistance of plants to Verticillium dahliae by enhancing callose deposition, promoting H2O2 accumulation and regulating hypersensitive response, and spraying VdSP8 protein on plants or introducing VdSP8 gene into plant cells to express VdSP8 protein in the plant body can significantly improve the resistance of the plants to Verticillium dahliae, and has good agricultural application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protection and crop disease control, and particularly relates to a Verticillium dahliae exocrine protein elicitor VdSP8, a coding gene thereof, and an application thereof. Background Art

[0002] Verticillium dahliae is a typical soil-borne vascular fungal pathogen with a broad host range and rich genetic variation. Its ability to persist in the environment as microsclerotia for long periods of time poses significant challenges to its control. Annually, this pathogen causes significant economic losses to global agriculture. Secreted proteins play a key role in V. dahliae's host infection. They are not only essential weapons for the pathogen's survival and pathogenicity, but also play a crucial role in the interaction between the pathogen and its host plant. Therefore, in-depth research on the functions of these secreted proteins, particularly those that elicit plant disease resistance, is of great scientific and practical value.

[0003] In the field of plant immunity research, elicitors, as an important immune regulatory factor, have been widely used to enhance plant resistance to pathogens. Elicitors are molecules produced by pathogens or other organisms that can trigger plant defense responses, thereby improving plant disease resistance. These molecules bind to receptors on plant cell membranes, activating a series of signal transduction pathways, leading to a series of disease resistance responses in plants, including callose deposition, accumulation of reactive oxygen species (ROS), and cell wall strengthening. Although a variety of elicitors have been studied and applied, many challenges remain in improving plant disease resistance. Therefore, the development of new and efficient elicitors and in-depth exploration of their mechanisms are of great significance for improving crop disease resistance, reducing the use of chemical pesticides, and promoting sustainable agricultural development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and provide a Verticillium dahliae exocrine protein elicitor VdSP8 and its application in enhancing plant disease resistance and improving growth performance.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A Verticillium dahliae exoprotein elicitor VdSP8, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] A gene encoding the Verticillium dahliae exoprotein elicitor VdSP8, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] The application also provides application of the Verticillium dahliae exoprotein elicitor VdSP8 or a coding gene of the Verticillium dahliae exoprotein elicitor VdSP8 in enhancing disease resistance of plants, wherein the enhancing disease resistance of plants comprises improving plant resistance and inducing plant defense response.

[0009] Preferably, the effective pH of the Verticillium dahliae exoprotein elicitor VdSP8 is 5-7.

[0010] Preferably, the improving plant resistance is improving plant resistance to Verticillium dahliae, Botrytis cinerea and Pseudomonas syringae; and the inducing plant defense response is inducing immune response of plants to Verticillium dahliae, Botrytis cinerea and Pseudomonas syringae and regulating cell death.

[0011] The application also provides application of the Verticillium dahliae exoprotein elicitor VdSP8 or a coding gene of the Verticillium dahliae exoprotein elicitor VdSP8 in improving growth performance of plants, wherein the improving growth performance of plants comprises improving plant growth rate, increasing fresh weight and promoting bolting of plants.

[0012] The application also provides a transgenic plant, wherein a VdSP8 gene is contained in a genome of the plant, so that the plant can express a VdSP8 protein.

[0013] The application also provides a method for enhancing disease resistance of plants or improving growth performance of plants, which comprises spraying a VdSP8 protein on a plant surface or introducing a VdSP8 gene into a plant cell to express the VdSP8 protein in the plant.

[0014] The application has the following remarkable technical effects:

[0015] 1. The application provides a new Verticillium dahliae exoprotein elicitor VdSP8 and application of a coding gene of the Verticillium dahliae exoprotein elicitor VdSP8 in enhancing disease resistance of plants and improving growth performance of plants, which can significantly improve resistance of host plants to various pathogenic bacteria, including Verticillium dahliae, Botrytis cinerea and Pseudomonas syringae, induce plant defense response, improve plant growth rate, increase fresh weight and promote bolting of plants, and has important agricultural application prospect.

[0016] 2、The research of the application shows that VdSP8 improves the resistance of plants to pathogenic bacteria through the pathways of enhancing callose deposition, promoting H2O2 accumulation, lignin accumulation, regulating SA (salicylic acid) and JA (jasmonic acid) related signal pathways and adjusting cell death, and spraying VdSP8 protein on plants or introducing VdSP8 gene into plant cells to make the plant express VdSP8 protein in the plant body can significantly improve the disease resistance of the plant, which has practical guiding significance in plant disease prevention and growth promotion applications.

[0017] The application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 For the screening results of VdSP8 transgenic Arabidopsis in Example 1, A is the molecular detection result, wherein M: DNA maker 2000, 1-8: transgenic line DNA template, 9: positive control, 10: wild type Arabidopsis Col-0; B is the Western blot detection result, wherein 1-8: total protein of transgenic line; 9: total protein of wild type Arabidopsis Col-0;

[0019] Figure 2 For the fresh weight change graph of VdSP8 transgenic Arabidopsis in Example 2;

[0020] Figure 3 For the bolting and plant height change graph of VdSP8 transgenic Arabidopsis in Example 2;

[0021] Figure 4 For the resistance graph of VdSP8 transgenic Arabidopsis to P. lageruginea in Example 3;

[0022] Figure 5 For the resistance graph of VdSP8 transgenic Arabidopsis to Botrytis cinerea in Example 3;

[0023] Figure 6 For the resistance graph of VdSP8 transgenic Arabidopsis to Pseudomonas syringae in Example 3;

[0024] Figure 7 For the callose deposition graph of VdSP8 transgenic Arabidopsis after inoculation with P. lageruginea in Example 4;

[0025] Figure 8 For the H2O2 accumulation graph of VdSP8 transgenic Arabidopsis after inoculation with P. lageruginea in Example 4;

[0026] Figure 9 For the HR change graph of VdSP8 transgenic Arabidopsis after inoculation with P. lageruginea in Example 4;

[0027] Figure 10This is a graph showing changes in lignin in VdSP8 transgenic Arabidopsis thaliana after inoculation with Verticillium dahliae in Example 4;

[0028] Figure 11 This is a diagram of protein purification of the protein elicitor VdSP8 in Example 5;

[0029] Figure 12 This is a diagram showing cell necrosis in tobacco and cotton induced by the protein elicitor VdSP8 in Example 5;

[0030] Figure 13 This is a diagram showing the enzyme activity determination of the protein elicitor VdSP8 at pH 3 to 8 in Example 5;

[0031] Figure 14 This is a graph showing changes in the expression of genes related to the SA and JA pathways after spraying VdSP8 protein on cotton in Example 6;

[0032] Figure 15 This is a graph showing changes in lignin content in cotton after spraying VdSP8 protein in Example 6; wherein, P: spraying protein VdSP8; NP: spraying PBS.

[0033] Figure 16 This is a graph showing the induction of Verticillium wilt resistance in cotton after spraying VdSP8 protein in Example 6. Here, -5: VdSP8 protein sprayed 5 days before inoculation; -2: VdSP8 protein sprayed 2 days before inoculation; +2: VdSP8 protein sprayed 2 days after inoculation; +5: VdSP8 protein sprayed 5 days after inoculation. DETAILED DESCRIPTION

[0034] The disclosed Verticillium dahliae exoprotein elicitor VdSP8 (hereinafter referred to as protein elicitor VdSP8) has an amino acid sequence as shown in SEQ ID NO. 1 in the sequence listing, and a gene encoding VdSP8, whose nucleotide sequence is shown in SEQ ID NO. 2 in the sequence listing. The following examples describe in detail how to utilize the VdSP8 protein or VdSP8 gene to enhance plant disease resistance and improve plant growth performance.

[0035] Example 1

[0036] Construction and screening of VdSP8 transgenic Arabidopsis thaliana.

[0037] 1. Test materials

[0038] The test plant was the wild type Col-0 of the Arabidopsis thaliana variety, which was obtained from the Crop Disease Monitoring and Control Team of the College of Plant Protection, Northwest Agriculture and Forestry University.

[0039] 2. Construction of VdSP8 transgenic Arabidopsis

[0040] A VdSP8 overexpression vector was constructed using the pCAMBIA 1300-Flag vector and transformed into Agrobacterium tumefaciens GV3101. Positive clones were selected and cultured in LB medium containing antibiotics. An inoculation solution containing 5% sucrose and 200 μL / L Silwet L-77 was prepared. The cells were collected by centrifugation and resuspended to an OD600 of 1.0 to obtain an Agrobacterium suspension. Inflorescences of Arabidopsis plants in the early fruiting stage were immersed in the Agrobacterium suspension for 20-30 seconds, ensuring that the leaves did not come into contact with the inoculation solution. After inoculation, the plants were placed in a dark box for 24 hours and then transferred to light conditions at 22-25°C for growth. Mature seeds (T0 generation) were collected after approximately three weeks.

[0041] 3. Screening and identification of VdSP8 transgenic Arabidopsis

[0042] The seeds were sterilized, treated with 75% alcohol and 10% sodium hypochlorite solution, and then washed with sterile water. The sterilized seeds were plated on 1 / 2MS solid medium containing Kana antibiotics and cultured at 4°C for 3 days before being transferred to 25°C under alternating light and dark conditions. After 14 days, plants with well-developed root systems were selected and transplanted into nutrient pots. For the initially screened plants, PCR amplification was performed, using wild-type Arabidopsis DNA as a negative control. The PCR products were detected by agarose gel. Plants with the correct bands were positive and continued to be cultured to the T3 generation. All positive homozygous lines that were green on the resistance medium were selected for protein identification. After protein extraction, Western blot detection was performed using anti-Flag antibodies.

[0043] The results are as follows Figure 1 As shown in A, no band was amplified from the wild-type Arabidopsis Col-0 genomic DNA, while bands of the same size as the positive control were amplified from the DNA of the eight candidate lines, indicating that VdSP8 insertion was identified in all eight lines. These overexpressing Arabidopsis were cultured to the T3 generation, and total protein was extracted and Western blot was used to detect the expression of VdSP8 protein. The results showed that VdSP8 protein was detected in OE-1, OE-2, OE-4, OE-5, OE-6, OE-7 and OE-8 (see Figure 1 B) Two VdSP8 transgenic Arabidopsis lines, OE-1 and OE-7, were selected for subsequent experiments and named OE-VdSP8#1 and OE-VdSP8#2, respectively.

[0044] Example 2

[0045] Growth phenotype analysis of VdSP8 transgenic Arabidopsis thaliana.

[0046] 1. Statistics of Arabidopsis seedling growth

[0047] VdSP8 transgenic Arabidopsis and wild-type seeds were sown on 1 / 2 MS medium. Arabidopsis seedlings grown on 1 / 2 MS medium for 10 days were removed from their roots and weighed for fresh weight. Ten seedlings from each strain were weighed, and the average fresh weight per seedling was calculated. This was repeated three times. The seedlings were then transplanted to nutrient soil for further growth. Arabidopsis plants grown for 20 and 30 days were removed from their roots and weighed for fresh weight. Ten seedlings from each strain were weighed, and the average fresh weight per seedling was calculated. This was repeated three times.

[0048] The results showed that at 30 days, the OE-VdSP8 Arabidopsis lines were significantly larger than the wild-type Arabidopsis (see Figure 2 A). The leaf weights of Arabidopsis seedlings were measured at 10, 20, and 30 days. It was found that the fresh weights of OE-VdSP8#1, OE-VdSP8#2, and wild-type Arabidopsis Col-0 seedlings were not significantly different at 10 and 20 days. At 30 days, the average fresh weights of OE-VdSP8#1 and OE-VdSP8#2 transgenic lines were 336.7 g and 319.3 g per seedling, respectively, which were 1.4-1.6 times the average fresh weight of wild-type Arabidopsis Col-0 seedlings (219.8 g) (see Figure 2). Figure 2 B).

[0049] 2. Arabidopsis bolting and plant height statistics after bolting

[0050] The bolting time of the Arabidopsis plants was recorded, and the plant height of the Arabidopsis plants after bolting was measured every 2 or 3 days until the growth was completed.

[0051] The results are as follows Figure 3 As shown in Figure A, VdSP8 transgenic Arabidopsis thaliana bolts earlier than wild-type Arabidopsis thaliana. The bolting time of VdSP8 transgenic plants is about 27 days, while the bolting time of wild-type Arabidopsis thaliana is about 28 days (see Figure 3 B). Monitoring the plant height of Arabidopsis after bolting revealed that the VdSP8 transgenic Arabidopsis plants were taller than wild-type Arabidopsis throughout the entire growth process after bolting. However, after the end of growth (i.e., about 50 days of growth), there was no significant difference in plant height between the OE-VdSP8 Arabidopsis line and the wild-type Arabidopsis (see Figure 3 C).

[0052] In summary, VdSP8 transgenic Arabidopsis overexpressing VdSP8 not only increased the leaf fresh weight of Arabidopsis seedlings, but also promoted the growth and development of Arabidopsis, prompted Arabidopsis plants to bolt earlier, and increased the plant height during the growth process of Arabidopsis.

[0053] Example 3

[0054] Identification of pathogen resistance of VdSP8 transgenic Arabidopsis thaliana.

[0055] 1. Experimental Materials

[0056] The Verticillium dahliae (V.dahliae) XJ592 strain, Botrytis cinerea (B.cinerea), and Pseudomonas syringae pv.tomato DC3000 (Pst DC3000) were derived from the Crop Disease Monitoring and Control Team of the College of Plant Protection, Northwest A&F University.

[0057] 2. Identification of resistance of VdSP8 transgenic Arabidopsis to Verticillium dahliae (Verticillium wilt pathogen)

[0058] The 3-week-old Arabidopsis thaliana was removed from the nutrient pot, the soil at the roots was gently shaken off, and the roots were rinsed with water. Then, the roots of all seedlings were completely immersed in the prepared spore suspension of Verticillium dahliae XJ592. Clean water was used as a control. After 20 minutes, the seedlings were taken out and transplanted into new sterile soil. They were placed in a constant temperature incubator with a light condition of 25±1℃ and a photoperiod of 14h light / 10h dark. After 15 days, the disease situation was observed and the disease index was calculated.

[0059] The results showed that there was no significant difference between VdSP8 transgenic Arabidopsis thaliana treated with water and wild type Arabidopsis thaliana (see Figure 4 A), and 15 days after inoculation with Verticillium dahliae, Arabidopsis thaliana showed typical symptoms of Verticillium wilt, such as yellowing and necrosis of rosette leaves, and the disease severity of wild-type Arabidopsis was significantly more severe than that of transgenic Arabidopsis thaliana overexpressing VdSP8 (see Figure 4 A). The statistical results of disease index and severity grade showed that wild-type Arabidopsis had more severe disease than transgenic Arabidopsis overexpressing VdSP8 (see Figure 4 Biomass statistical analysis showed that wild-type Arabidopsis was more sensitive to Verticillium dahliae than VdSP8 transgenic Arabidopsis (see Figure 4 D).

[0060] 3. Identification of resistance of VdSP8 transgenic Arabidopsis to Botrytis cinerea (gray mold)

[0061] The Botrytis cinerea stored at -80°C was inoculated on a PDA plate for activation. After 3 days, 3-4 pieces of the cake were placed in CM liquid culture medium and shaken at 25°C for about 3 days. The mycelium was removed by filtration with a filter cloth, and the spores were collected by centrifugation at 5000 rpm for 5 minutes. The spores were then prepared with sterile water to a concentration of 1×10 7A spore suspension of spores / mL was prepared for use. Four-week-old transgenic VdSP8 Arabidopsis and wild-type Arabidopsis (Col-0) were selected and the spore suspension of Botrytis cinerea was injected onto the underside of the rosette leaves until the entire leaf was filled. The inoculated Arabidopsis were placed in a humidifying box and incubated in a greenhouse at 25°C. Five days after injection, the disease was observed, photographed, the lesion diameter was counted, and the chlorophyll content of the Arabidopsis was measured.

[0062] The results showed that Arabidopsis thaliana showed different degrees of symptoms after inoculation with Botrytis cinerea. The leaves showed overall discoloration, rot and necrosis, and lesions formed at the inoculation site. The symptoms of Col-0 Arabidopsis thaliana were more obvious and the lesions were larger. In contrast, the symptoms of the OE-VdSP8 strain were milder (see Figure 5 A) The statistics of lesion area showed that the lesion area of ​​the infected Col-0 strain was approximately 44.6 mm 2 , 3.2 times larger than that of the OE-VdSP8 strain (see Figure 5 B). The chlorophyll content in plants tends to change with the growth conditions of the plants. The greater the damage caused by the disease, the faster the chlorophyll content decreases. Therefore, we sampled the diseased leaves and measured the chlorophyll content. The results showed that the total chlorophyll content, chlorophyll a content, and chlorophyll b content of wild-type Arabidopsis were significantly lower than those of VdSP8 transgenic Arabidopsis plants (see Figure 5 C).

[0063] 4. Identification of resistance of VdSP8 transgenic Arabidopsis to Pseudomonas syringae

[0064] To activate Pseudomonas syringae Pst DC3000, single colonies were selected and inoculated into LB liquid medium. The culture was shaken at 28°C for 1 day. The culture was centrifuged, the cells were harvested, and the cells were resuspended in 10mM MgCl2 solution to an OD600 of 0.004. The culture was then injected into the leaf until the entire leaf was completely filled. 48 hours after inoculation, the leaves were observed for disease and photographed. The diseased leaves were placed in a grinding tube containing small steel balls and ground using a tissue grinder. 1g of the sample was weighed and placed in a new centrifuge tube. 1mL of 10mM MgCl2 was added, and serial dilutions were made. The sample was then plated on LB plates and incubated at 28°C for 18 hours before colony counts were counted.

[0065] The results showed that Col-0 Arabidopsis was more susceptible to Pst DC3000 infection than the OE-VdSP8 line, while the OE-VdSP8 line showed stronger resistance to Pst DC3000 (see Figure 6A). In addition, we investigated the growth of the bacterium Pst DC3000 in plants inoculated with Col-0 and OE-VdSP8. In OE-VdSP8 plants infected with Pst DC3000, the growth of Pst DC3000 was inhibited, while more bacterial growth was observed in Col-0 plants infected with Pst DC3000 (see Figure 6 B).

[0066] In summary, overexpression of VdSP8 confers broader disease resistance to Arabidopsis thaliana, including the fungi Verticillium dahliae and Botrytis cinerea, and the bacterium Pseudomonas syringae.

[0067] Example 4

[0068] Analysis of the cytological mechanism of resistance to Verticillium wilt in VdSP8 transgenic Arabidopsis thaliana.

[0069] 1. Observation of callose deposition in VdSP8 transgenic Arabidopsis thaliana after inoculation with Verticillium dahliae

[0070] Callose deposition assay in Arabidopsis roots: Inoculation was performed using the root dipping method. Roots of Columbia wild-type Arabidopsis (Col-0) and VdSP8 transgenic Arabidopsis plants were excised 24 hours after inoculation. The roots were rinsed with water and then immersed in aniline blue dye in the dark for 2-4 hours. Finally, the roots were gently rinsed with water and slides were prepared. Fluorescence was observed and photographed using an FV3000 laser confocal microscope.

[0071] Arabidopsis leaf callose deposition assay: The in vitro leaf dorsal inoculation method was used. 4-5 week-old Columbia wild-type Arabidopsis (Col-0) and VdSP8 transgenic Arabidopsis were selected, with leaf widths approximately 1.0 cm. Carefully excise the leaves and place them in a Petri dish containing sterile filter paper moistened with sterile water, dorsal facing up. 10 μL of a prepared spore suspension of Verticillium dahliae XJ592 (concentration 1×10 7 Each treatment was performed in a dark incubator at 25°C (using sterile water as a negative control). At least 30 leaves were used for each treatment, and the experiment was repeated three times. 24 hours after inoculation, the leaves were decolorized in 75% ethanol to remove chlorophyll. The leaves were gently rinsed with water and then placed in aniline blue dye solution in the dark for 2-4 hours. The leaves were then rinsed with water and slides were prepared. Fluorescence was observed and photographed using an FV3000 laser confocal microscope.

[0072] The results showed that 24 hours after inoculation with Verticillium dahliae, more callose was deposited in the roots of VdSP8 transgenic Arabidopsis thaliana OE-VdSP8#1 and OE-VdSP8#2 compared with wild-type Arabidopsis Col-0 (see Figure 7 A). Arabidopsis thaliana treated with sterile water served as blank control, and no callose was produced (see Figure 7A). 24 hours after inoculation with Verticillium dahliae by detached leaf inoculation, OE-VdSP8#1 and OE-VdSP8#2 showed obvious callose deposition, while wild-type Arabidopsis showed less callose deposition (see Figure 7 B) These results indicate that overexpression of VdSP8 promotes the production of more callose in Arabidopsis thaliana infected with Verticillium dahliae, thereby enhancing disease resistance.

[0073] 2. H2O2 accumulation in VdSP8 transgenic Arabidopsis after inoculation with Verticillium dahliae

[0074] 48 hours after inoculation with Verticillium dahliae by detached leaf inoculation, the leaves were stained with DAB. The results showed that a large number of brown spots appeared in all Arabidopsis plants. The brown spots of VdSP8 transgenic Arabidopsis were larger than those of wild type. The Arabidopsis treated with sterile water served as a blank control. Except for the inoculation site, no brown spots were produced (see Figure 8 A). Using Image J to analyze the area of ​​brown spots, it was found that the area of ​​brown spots on the leaves of Col-0 Arabidopsis inoculated with Verticillium dahliae was about twice that of the uninoculated ones. The area of ​​brown spots on the leaves of VdSP8 transgenic Arabidopsis thaliana OE-VdSP8#1 and OE-VdSP8#2 inoculated with Verticillium dahliae was significantly larger than that of wild-type Arabidopsis Col-0 inoculated with Verticillium dahliae, which were 1.8 and 1.5 times that of wild-type Arabidopsis Col-0, respectively (see Figure 8 B) H2O2 content in Arabidopsis leaves inoculated with Verticillium dahliae was measured. A very low amount of H2O2 was detected in both Col-0 and VdSP8 transgenic Arabidopsis 0 hours after inoculation. 12 hours after inoculation, H2O2 content increased in both plants, with similar differences between them. 24 hours after inoculation, H2O2 levels increased in both Col-0 and VdSP8 transgenic Arabidopsis, reaching 8.28 μmol / g, 14.50 μmol / g, and 26.14 μmol / g, respectively. The rate of increase in H2O2 content in VdSP8 transgenic Arabidopsis was significantly faster than that in Col-0. 48 hours after inoculation, the H2O2 content in Col-0, OE-VdSP8#1 and OE-VdSP8#2 lines reached the maximum value, which was 11.94μmol / g, 27.50μmol / g and 33.43μmol / g, respectively. The H2O2 content in VdSP8 transgenic Arabidopsis was significantly higher than that in Col-0. After 72 hours and 96 hours of inoculation, the H2O2 content decreased, and there was no significant difference between VdSP8 transgenic Arabidopsis and Col-0 Arabidopsis (see Figure 8 C).

[0075] 3. Changes in the hypersensitive response (HR) of VdSP8 transgenic Arabidopsis thaliana after inoculation with Verticillium dahliae

[0076] To further verify the resistance of VdSP8 transgenic Arabidopsis to Verticillium wilt, we inoculated the leaves with Verticillium dahliae by detached leaf inoculation and stained the leaves with trypan blue 48 hours after inoculation. The results showed that necrotic cells were found at the inoculation site of both Col-0 and VdSP8 transgenic Arabidopsis 48 hours after inoculation with Verticillium dahliae. In addition, more cell death was observed in VdSP8 transgenic Arabidopsis, while only a small amount of cell death was found near the inoculation site in wild-type Arabidopsis (see Figure 9 ).

[0077] 4. Changes in lignin in VdSP8 transgenic Arabidopsis after inoculation with Verticillium dahliae

[0078] To further explore whether VdSP8 regulates lignin accumulation, we compared the total lignin content of VdSP8 transgenic Arabidopsis and Col-0 plants. The results showed that 2 days after inoculation with Verticillium dahliae, there was no significant difference in lignin content between Col-0 and OE-VdSP8 lines. However, 7 days after inoculation with Verticillium dahliae, lignin deposition in the OE-VdSP8 line increased rapidly and the lignin content was much higher than that of Col-0 (see Figure 10 ).

[0079] In summary, VdSP8 transgenic Arabidopsis significantly improved resistance to Verticillium wilt. Specifically, exogenous expression of VdSP8 promoted the production of callose and H2O2, while also increasing the HR response in Arabidopsis, thereby limiting hyphal expansion. Furthermore, after infection with Verticillium dahliae, VdSP8 further enhanced resistance to Verticillium wilt by regulating lignin accumulation.

[0080] Example 5

[0081] Functional study of protein elicitor VdSP8.

[0082] 1. VdSP8 ΔSP -His protein induction and purification

[0083] The prokaryotic expression vector pET28a was digested with EcoRI and VdSP8 was inserted into the vector by homologous recombination. ΔSP Ligated into the linearized pET28a vector to obtain VdSP8 ΔSP -His recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for prokaryotic expression. ΔSP-His monoclonal, inoculated into 5mL LB culture medium containing antibiotics (Kana), cultured at 37℃ overnight; took the overnight culture solution at a ratio of 1:100, inoculated into 100mL LB culture medium preheated to 37℃ and containing Kana, cultured for 3h or longer until the OD600 of the culture solution reached 0.4-0.8; added IPTG to a final concentration of 1mM, and the induction temperature and time were adjusted according to different proteins; collected the culture solution into a centrifuge tube, centrifuged at 4℃ 10000rpm for 10min, discarded the supernatant, collected the bacteria, and then entered the bacterial lysis step. Protein purification was performed using Ni-Sepharose. The results are as follows Figure 11 As shown, the protein concentration was the highest when eluted with 150 mM imidazole elution buffer 2.

[0084] 2. VdSP8 induces necrosis in tobacco cells

[0085] Transform GV3101 Agrobacterium with the vector containing VdSP8 and resuspend the cells in a medium containing 10 mM MgCl2, 0.2 mM acetosyringone, and 10 mM MES. Adjust the OD 600 To 0.4-0.5 (or adjust to 0.8-1.0 when co-transfected). Mix in a 1:1 ratio and incubate in the dark at 28°C for 1-3 hours. Use a syringe to inject the incubated Agrobacterium into the back of the tobacco leaves. After injection, keep moisturized in the dark at room temperature for 12 hours, and then culture in an incubator at 25±1°C, 16 hours of light / 8 hours of darkness. Observe and take pictures to record the necrosis of the tobacco leaves 5 days after injection, and count the proportion of necrotic leaves. The results are as follows Figure 12 As shown in A, VdSP8 can induce programmed necrosis in tobacco cells but cannot inhibit BAX-induced programmed necrosis in tobacco cells. Western blot results showed that GFP-VdSP8 fusion protein can be expressed in tobacco.

[0086] 3. VdSP8 induces necrosis in cotton cells

[0087] Use purified VdSP8 ΔSP -His protein was injected into different cotton cotyledons. After 3-5 days of injection, the phenotype of the cotton cotyledons was observed to see whether it triggered cell death and induced an immune response in cotton. Figure 12 As shown in B, the purified VdSP8 protein can induce cell death in cotton.

[0088] 4. Determination of protein elicitor VdSP8 enzyme activity

[0089] (1) Reagent preparation:

[0090] Tyrosine solution: Weigh 100 mg of tyrosine and dissolve it in 0.1 M hydrochloric acid to 100 mL. After sampling, dilute 10-fold with pure water.

[0091] Casein solution: Weigh 2 g of casein and dissolve it in a mixture of 0.2 M disodium hydrogen phosphate (61 mL) and 0.2 M sodium dihydrogen phosphate (39 mL).

[0092] Sodium carbonate solution: concentration 0.55 M. pH = 3-8 buffer: Prepared with 0.1 M citric acid and 0.2 M disodium hydrogen phosphate solution in proportion.

[0093] (2) Draw a standard curve: Take six test tubes, add different volumes of tyrosine solution (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mL), and make up to 1 mL with water. Add 5 mL of 0.55 M sodium carbonate solution and 1 mL of phenol reagent, shake well, and develop color in a 30°C water bath for 15 minutes. Use a spectrophotometer to measure the OD680 value and draw a standard curve. Figure 13 A.

[0094] (3) Enzyme activity determination: 2% casein was used as substrate, and 200 μL of enzyme activity detection system was prepared. The ratio of substrate to enzyme was 16:3:1, and the mixture was preheated at 30°C for 5 minutes. The OD440 value was measured using a microplate reader. 1 unit of enzyme activity (1U) was defined as the amount of subtilisin that hydrolyzed 1 μg of casein in 1 minute at 30°C and pH 7.5. The results were as follows: Figure 13 As shown in Figure B, the VdSP8 protein has enzymatic activity for hydrolyzing casein at pH = 5-7, and reaches a maximum of 63.69 U / mL at pH = 6, indicating that the VdSP8 protein is a neutral protease.

[0095] In summary, the protein elicitor VdSP8 can effectively induce necrotic responses in tobacco and cotton cells, and exhibits the highest neutral protease activity at pH 6.

[0096] Example 6

[0097] Application of protein elicitor VdSP8.

[0098] 1. Determination of the expression of SA (salicylic acid) and JA (jasmonic acid) signaling pathway-related genes and changes in lignin content after spraying VdSP8 protein aqueous solution

[0099] The results showed that after spraying the VdSP8 protein aqueous solution, the expression of SA signaling pathway-related genes GhPR2 and GhPR5 and JA signaling pathway-related gene GhPR4 was significantly upregulated. The expression levels of GhPR2 and GhPR4 reached their peak 48 hours after spraying the VdSP8 protein, while the peak expression level of GhPR5 appeared 24 hours after spraying the VdSP8 protein (see Figure 14On the second day after spraying the VdSP8 protein aqueous solution, the lignin content between the VdSP8 protein treated group and the control group began to differ, and the difference between the two reached a peak on the fifth day after spraying the VdSP8 protein, reaching 237.4 mg / g (see Figure 15 ).

[0100] 2. Effect of spraying VdSP8 protein aqueous solution on cotton resistance to Verticillium wilt

[0101] To further verify the role of VdSP8 protein in inducing plant disease resistance, we sprayed the purified VdSP8 protein onto cotton plants 5 days before inoculation, 2 days before inoculation, 2 days after inoculation, and 5 days after inoculation, respectively. The control group was sprayed with PBS solution. After inoculation, all cotton plants showed typical symptoms of Verticillium wilt, including yellowing and wilting of leaves, and obvious browning of vascular bundles (see Figure 16 A) The disease severity and disease index statistics showed that the cotton sprayed with VdSP8 protein two days before inoculation with Verticillium dahliae showed the lowest disease index of 60.83, which was 14.1% lower than the control group without protein spraying (see Figure 16 B and C).

[0102] In summary, spraying VdSP8 protein on plant surfaces may enhance host disease resistance by regulating the SA (salicylic acid) and JA (jasmonic acid) signaling pathways in cotton or directly affecting the lignin biosynthesis pathway. Furthermore, spraying VdSP8 protein two days before Verticillium dahliae infection was found to be most likely to induce resistance to Verticillium wilt in cotton plants, thereby reducing the incidence of Verticillium wilt, which has practical implications for agricultural applications.

[0103] The present invention provides a novel exoprotein elicitor, VdSP8, from Verticillium dahliae and its encoding gene for use in enhancing plant disease resistance and improving plant growth performance. The protein can effectively improve plant resistance to the pathogens Verticillium dahliae, Botrytis cinerea, and Pseudomonas syringae, induce plant defense responses, and increase plant growth rate, fresh weight, and bolting, thus possessing significant agricultural application prospects. An effective method for utilizing the VdSP8 protein or VdSP8 gene to enhance plant disease resistance and growth performance is to spray the VdSP8 protein onto the plant surface or to introduce the VdSP8 gene into plant cells to express the VdSP8 protein within the plant.

[0104] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A use of a Verticillium dahliae exoprotein elicitor VdSP8 or a gene encoding the Verticillium dahliae exoprotein elicitor VdSP8 in enhancing plant disease resistance, characterized in that: The amino acid sequence of the exoprotein elicitor VdSP8 of Verticillium dahliae is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the exoprotein elicitor VdSP8 of Verticillium dahliae is shown in SEQ ID NO.2; the enhancing plant disease resistance is to improve plant resistance and induce plant defense response; the improving plant resistance is to enhance plant resistance to Verticillium dahliae. Verticillium dahliae Botrytis cinerea Botrytis cinerea and / or Pseudomonas syringae Pseudomonas syringae The induction of plant defense response is to induce plants to Verticillium dahliae Verticillium dahliae Botrytis cinerea Botrytis cinerea and / or Pseudomonas syringae Pseudomonas syringae immune response and regulate cell death; the plant is Arabidopsis, tobacco or cotton.

2. A use of a Verticillium dahliae exoprotein elicitor VdSP8 or a gene encoding the Verticillium dahliae exoprotein elicitor VdSP8 in improving plant growth performance, characterized in that: The amino acid sequence of the Verticillium dahliae exoprotein elicitor VdSP8 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the Verticillium dahliae exoprotein elicitor VdSP8 is shown in SEQ ID NO.2; the improving plant growth performance is improving plant growth rate, increasing fresh weight and / or promoting plant bolting; the plant is Arabidopsis thaliana.

3. The use according to claim 1 or 2, characterized in that The VdSP8 protein is sprayed on the surface of the plant, or the VdSP8 gene is introduced into the plant cells to express the VdSP8 protein in the plant.

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