Application of an effector protein of tomato gray leaf spot pathogen in pest control

Abstract

The present invention provides an application of a tomato gray leaf spot pathogen effector protein in pest control, belonging to the field of peptide technology. The nucleotide sequence of the tomato gray leaf spot pathogen effector protein is shown in SEQ ID NO.7 in the sequence table; the tomato gray leaf spot pathogen is Stolonifera tomatoensis Tomato stem The pest is Bemisia tabaci. After the tomato seeds and tomato plants are treated with the effector protein solution of the present invention, Bemisia tabaci is not easy to feed on them, and the effect of repelling and controlling Bemisia tabaci is achieved.

Description

Technical Field

[0001] The invention relates to application of tomato gray leaf spot pathogen effector protein in pest control, belonging to the technical field of peptides. Background Art

[0002] Bemisia tabaci Bemisia tabaci Gennadius belongs to the family Aleyrodidae in the order Hemiptera. It is a highly omnivorous "super" pest that occurs worldwide. The host plants of Bemisia whitefly are as many as 600 species. It mainly causes damage by sucking plant sap, secreting honeydew to induce sooty mold, and spreading plant viruses. Among them, the disease caused by spreading plant viruses is the most serious, and it can spread more than 300 plant viruses. In recent years, with the rapid development of facility agriculture, small pests such as Bemisia whitefly have achieved year-round reproduction, seriously affecting the safety of vegetable production. At present, pest control in facility vegetable production is mainly based on chemical control, but the long-term, large-scale and irrational use of chemical pesticides has led to serious "3R" problems, such as high resistance of pests, rampant pests, especially pesticide residues, and possible environmental pollution and other public hazards. Therefore, it is very important to seek a technology that can replace traditional control measures.

[0003] There is no report in the prior art on using effector proteins to repel whiteflies. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application of a tomato gray leaf spot pathogen effector protein in pest control to achieve the following invention objectives: the screened tomato gray leaf spot pathogen effector protein has an obvious repellent effect on Bemisia tabaci.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] An application of a tomato gray leaf spot pathogen effector protein in pest control, wherein the nucleotide sequence of the tomato gray leaf spot pathogen effector protein is shown as SEQ ID NO.7 in the sequence table.

[0007] The tomato gray leaf spot pathogen is Stolonifera tomato.

[0008] The pest is Bemisia tabaci.

[0009] Compared with the prior art, the present invention achieves the following beneficial effects:

[0010] The present invention screened out 16 effector proteins whose expression was upregulated after tomato induction from the transcriptome of Stolonifera tomato, of which 7 effector proteins were upregulated at both 36h and 84h, and further screened out 2 effector proteins with little effect on the normal growth of Nicotiana benthamiana leaves, and finally screened out the effector protein Sts-950 that had an evasive effect on Bemisia tabaci.

[0011] After tomato seeds and tomato plants are treated with the effector protein Sts-950 solution of the present invention, whiteflies are not easy to feed on them, and the effect of repelling and controlling whiteflies is achieved. After the effector protein Sts-950 solution of the present invention is sprayed on tomato plants for 24 hours, whiteflies are released, and the repelling rates of whiteflies are 66.27% (6 hours after the release of whiteflies), 75.73% (12 hours after the release of whiteflies), and 83.11% (24 hours after the release of whiteflies). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a picture of the disease condition of tomato leaves 21 days after inoculation of tomato stolonifer SL1216;

[0013] Figure 2 This is a heat map of up-regulated effector proteins in the transcriptome information of Sclerotium SL1216;

[0014] Figure 3 This is a diagram of the growth status of tobacco leaves transiently expressing different effector proteins;

[0015] Figure 4 This is a Western blot detection image of effector proteins Sts950 and Sts408;

[0016] A is a Western blot detection image of effector protein Sts950, and B is a Western blot detection image of effector protein Sts408;

[0017] Figure 5 It is a bar graph of the selection rate of whitefly on tomato plants treated with effector protein Sts-950 solution;

[0018] Figure 6 The bar graph shows the selection rate of whitefly on tomato plants treated with effector protein Sts-408 solution. DETAILED DESCRIPTION

[0019] The tomato variety tested was Jinpeng No. 1, a tomato gray leaf spot susceptible variety;

[0020] The pathogen of tomato gray leaf spot used in the test was Stolonifera SL1216, which was preserved by the Green Control Laboratory of Vegetable Pests and Diseases of Weifang University of Science and Technology;

[0021] The phosphate buffer is 0.01 M PBS buffer at pH 7.4.

[0022] Example 1 Screening candidate effector proteins whose expression is upregulated after tomato induction from the transcriptome information of Sclerotium tomato

[0023] (1) Pathogenicity test of Sclerotium stoloniferum SL1216

[0024] After culturing the tomato spore fungus SL1216 on V8 medium, the spore solution was collected with sterile water and the spore concentration in the spore solution was adjusted to 1×10 5 The spore solution was sprayed on the back of the leaves of Jinpeng No. 1 tomatoes with 4 to 5 true leaves. Each tomato plant was sprayed with 500 μL. Each time, 12 tomatoes were sprayed. The experiment was repeated 3 times. 21 days after the spore solution was sprayed, the disease condition of the tomato leaves was observed. It was found that the lower leaves of the tomato began to become diseased, with brown spots and gray spots in the middle. The disease condition of the tomato plant leaves was 100%. Figure 1 .

[0025] (2) Transcriptome sequencing of tomato stolonifer induced by host leaves

[0026] Tomato Spores SL1216 were inoculated on V8 medium plates and cultured in the dark at 25°C for 6 days. The spores on the plates were washed with sterile water and 1 mL of 1×10 5 The spore liquid of each spore was inoculated into 100 mL of liquid PDA medium and cultured at 25°C and 160 rpm for 5 days to obtain mycelial balls.

[0027] Inoculate the mycelial pellets of Stolonifera tomato on the 2nd to 4th true leaves of tomatoes at the 5-6 leaf stage, spread the mycelial pellets evenly on the tomato leaves so that each mycelial pellet is in contact with the leaves, and collect samples together with the leaves and mycelial pellets at 36h and 84h after inoculation as samples. The uninoculated mycelial pellets (before inoculation) are used as control samples.

[0028] The RNA of the samples was extracted and reverse transcribed, and the transcriptome sequencing of Sclerotium SL1216 was performed. The transcriptome data were registered in the Chinese National Gene Bank database (https: / / www.cngb.org / index.html) with the registration number CNP0002533.

[0029] The transcriptome data analysis was performed using the genome sequencing information of the tomato strain CIDEFI-216 published by the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / nuccore / LGLR00000000.1) as the reference genome.

[0030] The assembled unigenes were annotated based on BLASTX searches against various protein databases, and the functional annotations of all unigenes were obtained using the GO database and the Blast2GO program.

[0031] The DESeq2 software package was used to analyze differentially expressed genes (DEGs). A total of 16 candidate effector proteins that were upregulated in the tomato stolonifer samples induced by tomato hosts were screened from the transcriptome information of tomato stolonifer SL1216, of which 7 were upregulated at both 36h and 84h (Table 1, Figure 2 as shown).

[0032] Table 1 Candidate effector proteins differentially expressed in Sclerotium tomato

[0033]

[0034] Example 2 Screening for candidate effector proteins that have little effect on the normal growth of Nicotiana benthamiana leaves

[0035] Seven candidate effector proteins KNG50493.1, KNG48427.1, KNG49655.1, KNG45810.1, KNG45830.1, KNG49619.1, and KNG50032.1, which were upregulated at 36 h and 84 h after induction, were selected and named Sts-168, Sts-690, Sts-829, Sts-556, Sts-408, Sts-792, and Sts-950, respectively. Their genes were cloned and transiently expressed in tobacco mediated by Agrobacterium, and the candidate effector proteins whose transient expression had less effect on the normal growth of Nicotiana benthamiana leaves were selected.

[0036] The specific method is as follows:

[0037] The total RNA of tomato sclerotium SL1216 was extracted according to the instructions of TransZol total RNA extraction kit (Beijing Quanshijin Biotechnology Co., Ltd., ET101), and then the RNA of tomato gray leaf spot pathogen was reverse transcribed using the reverse transcription kit (AE341-02) of Beijing Quanshijin Biotechnology Co., Ltd. to obtain cDNA.

[0038] Using the obtained cDNA as a template, PCR amplification was performed to obtain the target fragments of the coding genes of the above 7 effector proteins Sts-168, Sts-690, Sts-829, Sts-556, Sts-408, Sts-792 and Sts-950;

[0039] The nucleotide sequence of Sts-168 is shown in SEQ ID NO.1 in the sequence list;

[0040] The nucleotide sequence of Sts-690 is shown in SEQ ID NO.2 in the sequence list;

[0041] The nucleotide sequence of Sts-829 is shown in SEQ ID NO.3 in the sequence list;

[0042] The nucleotide sequence of Sts-556 is shown in SEQ ID NO.4 in the sequence list;

[0043] The nucleotide sequence of Sts-408 is shown in SEQ ID NO.5 in the sequence list;

[0044] The nucleotide sequence of Sts-792 is shown in SEQ ID NO.6 in the sequence list;

[0045] The nucleotide sequence of Sts-950 is shown as SEQ ID NO.7 in the sequence listing.

[0046] The target fragments were connected to the transient expression vector pYBA1133 by homologous recombination, transformed into Escherichia coli, and the positive transformants were screened by sequencing to obtain Escherichia coli containing the recombinant plasmid.

[0047] Escherichia coli containing the recombinant plasmid was cultured at 37°C and 220rpm, the recombinant plasmid was extracted, and the recombinant plasmid was transformed into Agrobacterium GV3101. After plating, the plate was inverted and cultured at 28°C for 2 days. A single clone of the positive transformant was picked and inoculated into LB medium and cultured at 28°C and 200rpm for 2 days. The bacterial liquid was centrifuged to collect the bacteria, resuspended with MMA (10 mM MgCl2, 10 mM MES, 0.15 mM AS), and the concentration was adjusted to OD600=1.0 to obtain a bacterial suspension. The bacterial suspension was infiltrated into Nicotiana benthamiana leaves with the help of a syringe for transient expression, and the transiently expressed INF (infestans host-specific elicitor) protein was used as a positive control.

[0048] The specific operation of infiltrating Nicotiana benthamiana leaves is as follows: for each Nicotiana benthamiana plant that has grown to the 4-5 leaf stage, select the 2nd and 3rd true leaves from the bottom, and use a syringe with the needle removed to inject the bacterial suspension into the inside of the leaf. Set one injection point on each leaf, and the injection volume at each injection point is 200 microliters.

[0049] After infiltrating the leaves of Nicotiana benthamiana, culture them in the dark for 12 hours, and then culture them normally for 6 days. Observe the changes of the leaves of Nicotiana benthamiana with the injection points and record them.

[0050] The results showed that the effector proteins to be tested were transiently expressed on Nicotiana benthamiana leaves through Agrobacterium-mediated expression. It was found that after infiltration of Nicotiana benthamiana leaves and normal culture for 6 days, tobacco leaves expressing INF protein (positive control) showed obvious local yellowing and cell necrosis. Tobacco leaves transiently expressing Sts-168, Sts-690, Sts-556, Sts-792 and Sts-829 also showed different degrees of yellowing, deformity or cell necrosis, while tobacco leaves expressing effector proteins Sts-950 and Sts-408 did not show yellowing, deformity or necrosis ( Figure 3 ), indicating that the expression of effector proteins Sts-950 and Sts-408 in tobacco cells does not affect the normal growth of tobacco cells. It is possible to consider applying this effector protein to tomatoes, which belong to the same Solanaceae family as tobacco, to study their effects on the ability of tomatoes to defend against diseases and pests.

[0051] Example 3 Construction of prokaryotic expression vector and acquisition of recombinant effector protein

[0052] The nucleotide sequences of effector proteins Sts-950 and Sts-408 of tomato gray leaf spot pathogen were cloned into the prokaryotic expression vector pET-28a(+) to obtain recombinant plasmids, which were transformed into Escherichia coli (DH5α), plated and incubated inverted at 37°C overnight, and the positive transformants were selected and sent to a sequencing company for sequencing verification.

[0053] The correctly connected recombinant plasmid was transformed into BL21 (DE3) competent cells, and after plating, the cells were inverted and cultured overnight at 37°C. A single colony of the positive transformant was picked and inoculated into 20 mL of LB medium. The cells were cultured overnight at 37°C and 220 rpm to obtain activated strains. The strains were inoculated into LB medium at a volume ratio of 1:100, and cultured at 220 rpm for 16 h. IPTG (final concentration of 0.5 mM) was added to the fermentation broth to induce the expression of the fusion protein. The bacterial liquid was centrifuged to collect the cells. After lysing the cells, the target protein was purified using the His-tag protein purification column BeyoGold™ His-tag Purification Resin (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). The protein concentration of the purified target protein was determined using a BCA kit (Nanjing Jiancheng). The concentrations of the two proteins Sts-950 and Sts-408 were 0.28 mg / mL and 0.30 mg / mL, respectively. SDS PAGE gel electrophoresis combined with Western blot detection of the above two prokaryotic expressed proteins showed that the band sizes of the Sts-950 and Sts-408 protein samples were approximately 13 KDa and 16 KDa (including the tagged protein), respectively, which were consistent with the expected protein size ( Figure 4 The obtained protein samples were stored at -20°C for future use.

[0054] Example 4 The repellent effect of the effector protein Sts-950 of tomato gray leaf spot pathogen on Bemisia tabaci

[0055] The effector protein Sts-950 solution obtained in Example 3 was diluted to 0.01 μmol / L and 0.05 μmol / L respectively using phosphate buffer;

[0056] A square insect cage with a size of 60.0 cm × 60.0 cm × 60.0 cm was made of 100-mesh insect-proof net and an aluminum alloy frame. An insect release port (Φ=2.5 cm) was made of 100-mesh insect-proof net in the center of the top to release whiteflies. It was tied with a rope when not in use.

[0057] The experiment was set up with treatment group and control group.

[0058] The treatment group was treated with effector protein Sts-950 solution, and the control group was treated with phosphate buffer solution.

[0059] The specific experimental process is:

[0060] (1) Seed soaking and germination

[0061] Soak the mature and plump tomato seeds (Jinpeng No. 1) in warm water at 55°C for about 15 minutes, and then soak the seeds in a solution of the effector protein Sts-950 at a concentration of 0.01μmol / L for another 8 hours. Then take out the seeds and place them in a culture dish filled with moist filter paper, moisten the filter paper with the above protein solution at 0.01μmol / L, and finally place the culture dish in a 25°C constant temperature box for germination under constant temperature and moisture conditions.

[0062] In the control group, seeds were soaked with phosphate buffer and filter paper was moistened with phosphate buffer.

[0063] (2) Colonization

[0064] After germination, the tomato seeds were sown in a 75-hole seedling tray filled with seedling medium and cultured in an intelligent greenhouse at 25-27°C. When the plants grew to the 3-leaf stage, the tomato plants were planted in pots (Φ=12cm). The planted tomato plants were cultured in an intelligent greenhouse at 25-27°C.

[0065] (3) Spraying effector protein

[0066] 28 days after planting, a solution of the effector protein Sts-950 with a concentration of 0.05 μmol / L was sprayed on the tomato leaves, with 2 mL of the protein solution sprayed on each tomato plant.

[0067] The control group was sprayed with phosphate buffer.

[0068] (4) Release of adult whiteflies

[0069] 24 hours after the tomato plants were sprayed with the effect protein solution or phosphate buffer, 100 newly emerged whiteflies were released from the insect release port in the treatment group and the control group, and the number of whiteflies landing on the leaves of the tomato plants was checked 6 hours, 12 hours and 24 hours after the release of the whiteflies. A total of 10 replicates were set, with 3 tomato plants in each replicate.

[0070] The formula for calculating the selection rate of whiteflies on tomato plants is: (number of whiteflies landing on tomato plant leaves / total number of insects) × 100%;

[0071] The calculation formula for the avoidance rate of the effector protein in the treatment group to Bemisia tabaci is: ((the number of Bemisia tabaci landing on the leaves of tomato plants in the control group - the number of Bemisia tabaci landing on the leaves of tomato plants in the treatment group) / the number of Bemisia tabaci landing on the leaves of tomato plants in the control group)) × 100%.

[0072] The selection rate results are as follows Figure 5 As shown in the figure, whiteflies were more inclined to feed on tomato plants in the control group. At 6h, 12h and 24h after the release of whitefly adults, the selection rates of whiteflies on tomato plants in the control group were 44%, 49.8% and 56.5%, respectively, which were significantly higher than the selection rates of 14.8%, 12% and 9.5% in the group treated with effector protein Sts-950 solution ( P=0.000 ).

[0073] The repellency rates of the effector protein Sts-950 solution on Bemisia whitefly were 66.27% (6h), 75.73% (12h) and 83.11% (24h), respectively; this indicates that the effector protein Sts-950 solution has the effect of repelling Bemisia whitefly and plays an effective role in controlling Bemisia whitefly on tomato plants.

[0074] Example 5 The repellent effect of the tomato gray leaf spot pathogenic fungus effector protein Sts-408 on Bemisia tabaci

[0075] The experiment was set up as an effector protein Sts-408 solution-treated group and a control group (treated with phosphate buffer).

[0076] The specific experimental process of the effector protein Sts-408 solution treatment group was the same as that of the effector protein Sts-950 solution treatment group in Example 4, with the only difference being that the effector protein Sts-408 solution was used to replace the effector protein Sts-950 solution; the number of whitefly adults released in both the treatment group and the control group was 80.

[0077] The results are as follows Figure 6As shown, the selection rates of whiteflies on tomato plants treated with effector protein Sts-408 solution at 6h, 12h and 24h were 28.1%, 27.1% and 26.6%, respectively, while the selection rates of whiteflies on tomato plants in the control group at 6h, 12h and 24h were 24.6%, 24.2% and 24.4%, respectively, with no significant difference ( P>0.05 ), indicating that the effector protein Sts-408 solution has no obvious repellent effect on whiteflies.

Claims

1. An application of an effector protein of tomato gray leaf spot pathogen in pest control, characterized in that: The nucleotide sequence of the tomato gray leaf spot pathogen effector protein is shown in SEQ ID NO.7 in the sequence table; the pest is Bemisia tabaci.

2. The use of a tomato gray leaf spot pathogen effector protein in pest control according to claim 1, characterized in that: The tomato gray leaf spot pathogen is Stolonifera tomato.

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