Effect of tomato leafminer oral secretion effector gene repat38 on inhibition of host plant defense

Abstract

The present application relates to the field of agricultural biotechnology, and particularly relates to the role of tomato leaf roller oral secretion effector gene REPAT38 in inhibiting host plant defense. The gene REPAT38 encodes a protein with an amino acid sequence as shown in SEQ ID NO: 2. The effector induces stomatal closure in 0.5h, and inhibits the synthesis of JA, ET and ABA in 1h. In addition, the synthesis of AOC, MYC2, ACS1A, PAL, PR1, EIL2 and SRK2E, 7 plant hormone response genes, are all inhibited at different time points. Therefore, the oral secretion effector REPAT38 can weaken the defense ability of tomato host, thereby improving the feeding performance of insects. When the gene is interfered, the ability of insects to inhibit plant defense is weakened.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically the role of the oral secretion effector gene REPAT38 of the tomato leafminer moth in suppressing host plant defense. Background Technology

[0002] In the long process of co-evolution with host plants, herbivorous insects have evolved various feeding methods to better adapt to and utilize their hosts. Simultaneously, plants have evolved various defense strategies to cope with the feeding of herbivorous insects, including direct and indirect defense. Insects release saliva during feeding, which comes into direct contact with the plant. The components of this saliva are recognized by the host plant and can induce or inhibit a defensive response. The components that can be recognized by the plant and induce a defensive response are called elicitors, while the components that inhibit a plant's defensive response are called effectors.

[0003] The tomato leafminer, Phthorimaea absoluta, is an omnivorous pest that can damage nearly 40 plant species. Therefore, it is necessary to study the interaction between the oral secretions of the tomato leafminer and the defense response of tomato plants. Summary of the Invention

[0004] The purpose of this invention is to provide an effector of oral secretions from the tomato leafminer moth.

[0005] Another object of the present invention is to provide the above-mentioned oral secretion effector gene REPAT38 of the tomato leafminer moth.

[0006] Another object of the present invention is to provide the application of the above-mentioned gene in host plants for the defense against tomato leafminer.

[0007] Another object of the present invention is to provide a method for improving the defense capabilities of host plants of the tomato leafminer.

[0008] According to a specific embodiment of the present invention, the oral secretion effector gene REPAT38 of the tomato leafminer moth was cloned for the first time, and its full-length cDNA nucleotide sequence is shown in SEQ ID No:1.

[0009] SEQ ID No:1:

[0010] GAACTGTGATAAGTTGCTAACATTTTAAAACATAAAGATGAAGGCCATCCTAGTACTATGCATGTTGGCAGCGCTGGCTCTTACCGAAGCATCAGTTGCTCGTGCCCCGGCAACACGAAGCAATCCATCTTTGGGATACATTCAGTATGGAGATAGACTCCTAGCAAGGCAGTATCTTGCCAAGGGAGCGCGACCAAACGCCATCCA GTATCGAGACCTGATTTACCGCGGGAACGCTACCACGCGAATCAGTGCCATTCAAGCCATGGAAGTGGGCTACACGCAGTGGGCCAGCCGTGGGTCGTAGCTGGTGGAATTGGTTGGAACTCGGCCACAGTGCGCGTGCAATCCGCGCGGCTACGGCTATAACTATCAGATCGACATTTGGGGACGATGAGTTACTACATATT.

[0011] The amino acid sequence of the oral secretion effector receptor REPAT38 of the tomato leafminer is shown in SEQ ID NO: 2.

[0012] SEQ ID NO: 2:

[0013] MKAILVLCMLAALALTEASVARAPATRSNPSLGYIQYGDRLLARQYLAKGAR PNAIQYRDLIYRGNATTRISAIQAMEVGYTQWASAWVVAGGIGWNSATVRVQSAR GYGYNYQIDIWGR.

[0014] The method for enhancing the defense capabilities of the host plant of the tomato leafminer according to the present invention includes the step of interfering with the expression of the oral secretion effector gene REPAT38 of the tomato leafminer, wherein the oral secretion effector gene REPAT38 encodes a protein with the amino acid sequence shown in SEQ ID NO:2.

[0015] According to the method for improving the defense capabilities of the host plant of the tomato leafminer according to this application, the tomato leafminer is fed dsRNA of the oral secretion effector gene REPAT38.

[0016] According to the method for enhancing the defense capabilities of tomato leafminer host plants disclosed in this application, the dsRNA is obtained by amplifying the oral secretion effector gene REPAT38 of the tomato leafminer using the following primers.

[0017] dsREPAT38F:5′ TAATACGACTCACTATAGGG GGCAACACGAAGCAATCCAT3′;

[0018] dsREPAT38R: 5′ TAATACGACTCACTATAGGG ATTCCACCAGCTACGACCCA3′.

[0019] This invention provides the application of the oral secretion effector REPAT38 of the tomato leafminer in suppressing host plant defenses. The results showed that interfering with the REPAT38 gene significantly increased stomatal conductance in tomato leaves compared to the control group, indicating that the REPAT38 effector can induce stomatal closure. Interfering with the REPAT38 gene also resulted in higher synthesis levels of JA, ET, and SA compared to the control, indicating that the REPAT38 gene can inhibit the synthesis of JA, ET, and SA. Furthermore, interfering with the REPAT38 gene resulted in higher expression levels of seven plant hormone response genes (AOC, MYC2, ACS1A, PAL, PR1, EIL2, and SRK2E) at different time points compared to the control, indicating that the REPAT38 gene can suppress their expression. Therefore, the oral secretion effector REPAT38 can weaken the defense capabilities of the tomato host, thereby improving the insect's feeding performance. Interfering with this gene weakens the insect's ability to suppress plant defenses. Attached Figure Description

[0020] Figure 1 The structure of the oral secretion effector REPAT38 of the tomato leafminer moth is shown;

[0021] Figure 2 This shows the change in the expression level of the REPAT38 gene dsRNA after the tomato leafminer feeds on it.

[0022] Figure 3 This shows the effect of REPAT38 on stomatal opening and closing in the host plant, tomato leaves;

[0023] Figure 4 This demonstrates the effect of REPAT38 on regulating plant hormone synthesis;

[0024] Figure 5 This demonstrates the effect of REPAT38 on the regulation of plant response gene expression. Detailed Implementation

[0025] Example 1: Cloning of the full-length cDNA sequence of the REPAT38 gene of the tomato leafminer.

[0026] Four tomato leafminer larvae were placed in 1.5 mL centrifuge tubes, frozen in liquid nitrogen, and then ground into a paste using a grinder. RNA was extracted and stored at -80°C for later use. Following the instructions of the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for PCR kit, cDNA was synthesized from the extracted RNA. Primers were designed using the cDNA as a template for PCR amplification. The designed primers are shown in Table 1.

[0027] Table 1 Primer sequences for cloning the full-length cDNA of the REPAT38 gene.

[0028]

[0029] Using the sequences in Table 1, the full-length cDNA sequence of the REPAT38 gene was obtained by PCR amplification. The resulting gene has the nucleotide sequence shown in SEQ ID No:1, encoding 120 amino acids as shown in SEQ ID No:2. Analysis of the amino acid sequence encoded by the cloned gene showed a calculated molecular weight of 13.17 kDa and a theoretical isoelectric point (pI) of 10.07. Figure 1 As shown, REPAT38 is a small secretory protein with 120 amino acid residues. Its N-terminal region contains a signal peptide, which is cleaved at the 18-19 amino acid position and contains the transcription activator MBF2.

[0030] Example 2: Analysis of the response of the REPAT38 gene to host plant defense against tomato leafminer.

[0031] 2.1 Synthesis of target gene dsRNA and related gene primers

[0032] (1) Design the REPAT38 gene dsRNA, quantitative PCR of 12 plant hormone response genes (AOC, ACO1, MYC2, ACS1A, ACS1B, AOS, OPR3, PAL, PR1, EIL2, ERF1 and SRK2E), and primer sequences of the internal reference gene β-actin, as shown in Table 2 below.

[0033] Table 2. Relevant primer sequences

[0034]

[0035]

[0036] Total RNA extraction and cDNA synthesis for the tomato leafminer were performed as in Example 1. T7 primer PCR amplification and product purification were conducted, with the purified PCR product serving as the template for dsRNA synthesis. dsRNA was synthesized and purified using a kit, following the kit's instructions. Total RNA extraction and cDNA synthesis were performed according to the instructions for the plant RNA extraction and cDNA synthesis kit.

[0037] 2.2dsRNA feeding

[0038] Fresh leaves were dried for 2 hours. Simultaneously, second-instar larvae of the tomato leafminer were collected and starved for 2 hours. The larvae were then allowed to fully absorb dsREPAT38 at a concentration of 25 μg / mL through the petiole. After feeding on the leaves for 48 hours, samples were collected. Tomato leafminers fed an aqueous solution containing dsEGFP (final dsEGFP concentration of 25 μg / mL) served as a control. Five replicates were performed in each control and treatment group, with 30 larvae per replicate. The quantitative PCR detection kit was followed according to the instructions. -ΔΔCT The relative expression level of the dsREPAT38 gene after RNAi interference was calculated. The silencing efficiency of REPAT38 was detected after 60 h of feeding. It was found that the expression level of REPAT38 gene in the dsREPAT38 feeding group was significantly downregulated, which was significantly reduced by 56.7% compared with the control group (t=2.571, df=8, P<0.05).

[0039] 2.3 Observation of stomata on the lower epidermis of leaves

[0040] Lower leaves of tomato plants were selected. Larvae treated with dsEGFP and dsREPAT38 fed on the leaves for 0.5 h, 1 h, 2 h, and 4 h, respectively. The blank control group consisted of leaves that had not been fed by larvae. Approximately 1 square centimeter of lower epidermis was removed from near the wound on the leaf with a scalpel. The lower epidermis was placed on a glass slide, covered with a coverslip, and immediately observed under a biological microscope (BX63). Three fields of view were randomly selected from each sample, and the number of open and closed stomata was counted, and the stomatal closure rate was calculated. The results showed that at 0.5 h, the stomatal conductance of tomato leaves increased significantly by 31.07% (OS) compared to the control group. dsREPAT38 vs.OS dsEGFP At 1 hour, stomatal conductance decreased significantly by 22.36%, indicating that the oral secretion effector REPAT38 of the tomato leafminer regulates stomatal conductance in tomato leaves. Figure 3 As shown.

[0041] 2.4 Detection of plant hormone content

[0042] A one-step sandwich enzyme-linked immunosorbent assay (ELISA) using double antibodies was employed. Samples, standards, and HRP-labeled detection antibodies were sequentially added to microwells pre-coated with antibodies against jasmonic acid (JA), jasmonicyl L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA). After incubation and thorough washing, the sample was developed using the substrate TMB. TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the concentrations of jasmonic acid (JA), jasmonicyl L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the sample concentration was calculated. Figure 4 As shown, this effector inhibits the synthesis of JA, ET and SA in 1 hour.

[0043] 2.5 Detection of plant response gene expression

[0044] Follow the instructions for the quantitative PCR detection kit, and proceed with the test using 2... -ΔΔCT The method calculates the relative expression level of genes. For example... Figure 5 As shown, seven plant hormone response genes, namely AOC, MYC2, ACS1A, PAL, PR1, EIL2, and SRK2E, were suppressed at different time points.

[0045] The above research results indicate that REPAT38, as an oral secretion effector, can weaken the defense capabilities of the tomato host and plays an important role in the process of the tomato leafminer feeding on tomato leaves.

[0046] This invention cloned the full-length cDNA of the oral secretion effector REPAT38 gene from the tomato leafminer. Using RNAi technology, targeting the oral secretion effector REPAT38 gene, changes in leaf stomatal activity, plant hormone concentrations (jasmonic acid (JA), jasmonicyl L-isoleucine (JA-Ile), salicylic acid (SA), ethylene (ET), and abscisic acid (ABA)) and the relative expression levels of 12 plant hormone response genes (AOC, ACO1, MYC2, ACS1A, ACS1B, AOS, OPR3, PAL, PR1, EIL2, ERF1, and SRK2E) were monitored at 0.5, 1, 2, and 4 h. The molecular mechanism of REPAT38-mediated plant-insect interaction was explored, showing that:

[0047] When the REPAT38 gene was interfered with, the stomatal conductance of tomato leaves increased significantly at 0.5 h compared with the control group, indicating that the effector REPAT38 gene can induce stomatal closure.

[0048] When the REPAT38 gene was interfered with, the synthesis of JA, ET and SA was higher than that of the control, indicating that the REPAT38 gene can inhibit the synthesis of JA, ET and SA.

[0049] When the REPAT38 gene was interfered with, the expression levels of seven plant hormone response genes, namely AOC, MYC2, ACS1A, PAL, PR1, EIL2, and SRK2E, were higher than those of the control at different time points, indicating that the REPAT38 gene can inhibit the expression of this gene.

[0050] This indicates that the oral secretion effector REPAT38 can weaken the defense capabilities of the tomato host, thereby enhancing the feeding performance of insects. When this gene is interfered with, the insect's ability to suppress plant defenses is weakened, as reflected in changes in plant defense hormones and response genes. Plant hormones such as jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) are involved in the regulation of herbivorous defenses. In biological stress reprogramming, transcription factors coordinate the biosynthesis of defense genes and the dynamic interactions between metabolites during biosynthesis.

[0051] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. The oral secretion effector gene REPAT38 of the tomato leafminer moth, characterized in that, The oral secretion effector gene REPAT38 of the tomato leafminer moth encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

2. The tomato leafminer moth oral secretion effector gene REPAT38 according to claim 1, characterized in that, The nucleotide sequence of the gene REPAT38 is shown in SEQ ID No:

1.

3. A method for weakening the defense capabilities of tomato leafminer moths against host plants, characterized in that, Interference with the expression of the oral secretion effector gene REPAT38 of the tomato leafminer moth, wherein the oral secretion effector gene REPAT38 encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

4. The method for weakening the defense capabilities of the tomato leafminer against its host plant according to claim 3, characterized in that, The tomato leafminer was fed the dsRNA of the oral secretion effector gene REPAT38.

5. The method for weakening the defense capabilities of the tomato leafminer against its host plant according to claim 3, characterized in that, dsRNA was obtained by amplifying the oral secretion effector gene REPAT38 of the tomato leafminer using the following primers. dsREPAT38 F:5´ TAATACGACTCACTATAGGG GGCAACACGAAGCAATCCAT3´; dsREPAT38 R:5´ TAATACGACTCACTATAGGG ATTCCACCAGCTACGACCCA3´。

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