Diaphorina citri effect protein DcPDI1 and application thereof

Abstract

The invention discloses a diaphorina citri effector protein DcPDI1 and an application thereof. The amino acid sequence of the diaphorina citri effector protein DcPDI1 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the diaphorina citri effector protein DcPDI1 is as shown in SEQ ID NO. 2. After the diaphorina citri DcPDI1 is knocked out, the egg laying amount and the developmental duration of the diaphorina citri are remarkably reduced, and it is indicated that the DcPDI1 plays an important role in growth and development of the diaphorina citri. The diaphorina citri effect protein DcPDI1 can be a new molecular target spot, plays a key role in activating defensive response of host plants and helping diaphorina citri to eat, and provides a new insight for a molecular mechanism of interaction between diaphorina citri and plants.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to a citrus psyllid effector protein DcPDI1 and its applications. Background Technology

[0002] Citrus psyllid ( Diaphorina citri Citrus psyllids (Kuwayama) belong to the family Psyllidaceae in the order Hemiptera. Currently, control of citrus psyllids mainly relies on chemical pesticides, but long-term use of these pesticides has led to pesticide resistance. Therefore, finding new strategies for controlling citrus psyllids is crucial.

[0003] Herbivorous insects secrete saliva into their host plants during feeding. The components of this saliva play a crucial role in the insects' plant consumption, not only pre-digesting and interpreting the nutrients in the ingested plant, but also potentially containing effector proteins—components involved in the early defense responses of the host plant. The citrus psyllid is a piercing-sucking insect that feeds on the phloem sap of plants, injecting saliva carrying the pathogen of Huanglongbing (HLB) into the plant during feeding. Identifying potential effector protein genes in the citrus psyllid will help understand the mechanisms by which it overcomes host plant defenses and contribute to the development of novel plant immunomodulators for the control of the citrus psyllid. Summary of the Invention

[0004] To screen for potential effector proteins in the citrus psyllid genome, we analyzed LC-MS / MS data of leaves from citrus psyllids feeding on sweet oranges and identified a gene encoding an effector protein, named DcPDI1. To investigate the function of DcPDI1, we constructed a vector to introduce DcPDI1 into *E. coli* and transformed it into *Agrobacterium*. Injection into leaves of *Nicotiana benthamiana* that had grown for four weeks revealed that this protein induced programmed cell death (PCD). Significant changes in the expression of key genes related to defense pathways were observed in tobacco plants overexpressing DcPDI1, indicating that this protein plays a crucial role in regulating plant defense responses.

[0005] The first objective of this invention is to provide a citrus psyllid effector protein DcPDI1, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] A second object of the present invention is to provide a gene encoding the aforementioned citrus psyllid effector protein DcPDI1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] A third objective of this invention is to provide the application of the aforementioned citrus psyllid effector protein DcPDI1 or the aforementioned gene in the control of citrus psyllids.

[0008] Preferably, it is the application of a preparation that silences the gene of the citrus psyllid effector protein DcPDI1 in the control of citrus psyllids.

[0009] Preferably, the formulation is the dsRNA of the gene encoding the citrus psyllid effector protein DcPDI1.

[0010] Preferably, the control of citrus psyllids involves reducing the number of eggs laid and the development period of citrus psyllids.

[0011] A fourth objective of this invention is to provide the use of preparations that inhibit the above-mentioned citrus psyllid effector protein DcPDI1 or inhibit the above-mentioned gene in the preparation of products for the prevention and control of citrus psyllids.

[0012] The fifth objective of this invention is to provide the application of the aforementioned citrus psyllid effector protein DcPDI1 in regulating plant defense responses.

[0013] Preferably, the application is achieved by inhibiting the promotion of plant ROS by the citrus psyllid effector protein DcPDI1, inhibiting plant salicylic acid pathway-related genes, and / or promoting jasmonic acid pathway-related genes.

[0014] Preferably, the salicylic acid pathway-related genes are NPR1, PAL, and PR1a, the jasmonic acid pathway-related genes are PR3, PR4, LOX2, MYC2, and JIP21, and the plant is tobacco.

[0015] Advantages of this invention: This invention investigates the function of DcPDI1 in the citrus psyllid, demonstrating that knockdown of DcPDI1 significantly reduces oviposition and developmental duration, indicating that DcPDI1 plays a crucial role in the growth and development of the citrus psyllid. The citrus psyllid effector protein DcPDI1 may be a novel molecular target, playing a key role in activating host plant defense responses and aiding in feeding, providing new insights into the molecular mechanisms of citrus psyllid-plant interactions. Attached Figure Description

[0016] Figure 1 The citrus psyllid effector protein DcPDI1 induces necrosis in tobacco leaves.

[0017] Figure 2 This describes the changes in H2O2 levels in tobacco leaves after DcPDI1 injection.

[0018] Figure 3 This is the sequence information of the citrus psyllid effector protein DcPDI1.

[0019] Figure 4 This describes the expression of defense-related genes in tobacco after DcPDI injection.

[0020] Figure 5 It is a citrus psyllid. DcPDI1 Gene expression at different ages.

[0021] Figure 6 It is a citrus psyllid. DcPDI1 Gene expression in different tissues.

[0022] Figure 7 It is a citrus psyllid. DcPDI1 Gene expression before and after feeding.

[0023] Figure 8 This is the function of DcPDI1 in citrus psyllids. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Example: 1. LC-MS / MS analysis of saliva from citrus psyllids Citrus psyllids and sweet orange plants are raised in the greenhouse of the Fruit Tree Research Institute of Guangdong Academy of Agricultural Sciences.

[0026] Two-month-old sweet orange seedlings were placed in 15 cm x 15 cm x 15 cm nylon mesh cages. Fifty citrus psyllids of different ages were starved for 2 hours, then placed in the cages to feed on sweet orange leaves for 48 hours. Afterward, all citrus psyllids were removed, the area was cleaned, and the fed sweet orange leaves were collected, wrapped in aluminum foil, and treated with liquid nitrogen. The samples were then sent to Shanghai Zhongke New Life Biotechnology Co., Ltd. for LC-MS / MS analysis.

[0027] 2. Screening for candidate effector proteins of citrus psyllid After LC-MS / MS sequencing of sweet orange leaves, reference genomes of sweet orange and citrus psyllids were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ). Following alignment, sweet orange gene data from the original sequencing data were filtered out, and the remaining data was analyzed to obtain candidate salivary proteins for the citrus psyllid. Signal peptide and transmembrane domain predictions were performed on all candidate proteins, retaining those with signal peptides but no transmembrane domains as candidate effector proteins.

[0028] LC-MS / MS analysis predicted 32 potential citrus psyllid salivary effector proteins.

[0029] 3. Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA. The experimental procedures were performed according to the kit instructions. The RNA extraction kit (DP451) and reverse transcription kit (KR126-02) were both purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0030] 4. Cloning potential effector proteins Based on the obtained candidate effector protein gene sequences, specific primers were designed using the NCBI database Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), and the primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. (Table 1). PCR was performed using citrus psyllid cDNA as a template. High-fidelity enzymes were purchased from Nanjing Novizan Biotechnology Co., Ltd. The PCR amplification system was as follows: 2 × Phanta Max Master Mix 25 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA 2 μL, ddH2O 19 μL. The PCR amplification program was as follows: pre-denaturation 95℃ for 3 min, denaturation 95℃ for 15 sec, annealing 55℃ for 15 sec, extension 72℃ for 15 sec, 34 cycles, and a final extension 72℃ for 7 min. After PCR, the amplification products were electrophoresed on a 1% agarose gel, the gel strips were cut off, and the gel was recovered using a gel extraction kit. The gel extraction kit (DP204) was purchased from Tiangen Biotech (Beijing) Co., Ltd. The extracted gel products were sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the predicted sequences to obtain the full-length cDNA of the effector protein genes.

[0031] By designing specific primers, 27 complete open read frames (ORFs) were cloned using citrus psyllid cDNA as a template.

[0032] Table 1. PCR primer sequences for 27 candidate citrus psyllid effector protein genes. ; .

[0033] Lowercase letter bases represent homologous arm sequences.

[0034] 5. Construction of candidate effector protein expression vectors The homologous recombination reagent (C115-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd. The reaction system was as follows (operated on ice): 5 μL of 2 × ClonExpress Mix, 1 μL of linearized pCAMBIA1300-EGFP vector, 1 μL of insert fragment, and 3 μL of ddH2O. The centrifuge tubes containing the reaction solution were incubated at 50°C for 15 min. The pCAMBIA1300-EGFP vector (V013557) was purchased from Shanghai Newp Biotechnology Co., Ltd. The restriction enzymes were purchased from NEB, with restriction sites of BamHI (R0136V) and XbaI (R0145V). The reaction solution from the previous step was added to 100 μL of DH5α competent cells, incubated on ice for 30 min, then incubated at 42°C for 45 sec, and then incubated on ice for 3 min. 700 μL of antibiotic-free LB liquid was added, and the mixture was incubated at 37°C for 1 h using a shaker at 200 rpm.

[0035] Centrifuge the reaction solution from the previous step at 5000 rpm for 3 min, discard 700 μL of supernatant, resuspend the remaining liquid, and gently spread it onto LB agar plates containing cannabinoids (concentration of 50 μg / mL) using a sterile spreader. Incubate the plates upside down in a 37°C incubator for 15 h.

[0036] Positive clones were selected and sequenced. The sequencing results were compared with the predicted sequences to obtain the recombinant expression plasmids for the effector proteins. Plasmid extraction was performed according to the kit instructions. The plasmid extraction kit (P1001-02) was purchased from Shanghai Maigen Biotechnology Co., Ltd.

[0037] Expression vectors for 27 candidate effector protein genes were successfully constructed.

[0038] The recombinant expression plasmid was transformed into Agrobacterium GV3101. The main steps are as follows: 0.5 μg of the recombinant plasmid was added to GV3101 competent cells, and the cells were incubated on ice for 5 min, in liquid nitrogen for 5 min, at 37℃ for 5 min, and on ice for 5 min. 700 μL of antibiotic-free LB broth was added, and the cells were incubated in a shaker at 28℃ for 2 h. GV3101 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0039] Centrifuge the reaction solution from the previous step at 5000 rpm for 3 min, discard 700 μL of supernatant, resuspend the remaining liquid, and spread it onto LB agar plates containing Cannabidiol (concentration 50 μg / mL). Incubate the plates upside down in a 28℃ incubator for 24 h.

[0040] Select positive clone strains for culture PCR (using a single clone strain as a template, the PCR system, procedure and primers are as in step 4) to obtain Agrobacterium containing recombinant plasmids of effector proteins.

[0041] 6. Transient expression of candidate effector protein genes in Nicotiana benthamiana Agrobacterium containing the recombinant plasmid of the effector protein was cultured in 50 mL of LB liquid medium containing Kanamycin (50 μg / mL) at 28°C for approximately 24 h. The cells were collected by centrifugation at 5000 rpm for 10 min, and resuspended in a pre-prepared infection solution (10 mM MES, 10 mM MgCl2). Then, 100 μM acetylsyringone (AS) was added. 600 =0.5-1.0, let stand at room temperature for 2 h. Inject the bacterial solution into the leaves of tobacco plants that have grown for 4 weeks using a 1 mL disposable syringe, injecting 5 sites on each leaf, repeating the injection for 3-5 tobacco plants, injecting 4 leaves per plant. Use Phytophthora infestans ( Phytophthora infestans elicitin (INF1, GenBank: AAV92919.1) and apoptosis regulator (BCL2 associated X, BaX, GenBank: JAAKGM020000011.1) were used as positive controls, and green fluorescent protein (GFP) was used as a negative control.

[0042] Twenty-seven candidate effector proteins were injected into Nicotiana benthamiana cultured for four weeks, with five injection sites per leaf. The results showed that 36 hours after injection, one candidate effector protein (named DcPDI1) induced a necrotic phenotype in the tobacco leaves. Necrotic spots were observed after staining with 3,3'-diaminobenzidine (DAB) and destaining with anhydrous ethanol, indicating that DcPDI1 induced necrosis in tobacco leaves accompanied by the production of reactive oxygen species (ROS). Even after removing the signal peptide sequence of DcPDI1 (MRRLSLLFLLISPLVVFA), it still induced necrosis in tobacco leaves. Figure 1 By detecting the changes in H2O2 content in tobacco leaves after DcPDI1 injection, it was found that the H2O2 level in leaves injected with DcPDI1 was significantly higher than that in leaves injected without DcPDI1, and the H2O2 level reached its peak on days 4-5. Figure 2 The results indicate that DcPDI1 promotes the accumulation of ROS in tobacco.

[0043] Using specific primers (121346 in Table 1), the complete open reading frame (ORF) of DcPDI1 was amplified, with a length of 1494 bp. The sequence is as follows: Figure 3 As shown, the nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1.

[0044] The physicochemical properties of the DcPDI1 sequence were analyzed using Expasy software (https: / / www.expasy.org / ) (Table 2). The molecular weight of this gene protein is 55.8 kDa, the isoelectric point is 4.89, the instability coefficient is 37.88, the aliphatic amino acid index is 88.87, and the total hydrophilicity is -0.299, indicating that it is a hydrophilic protein.

[0045] Table 2 Physicochemical properties of the DcPDI1 sequence .

[0046] 7. Expression of key genes in defense-related pathways in tobacco after DcPDI1 injection To verify whether DcPDI1 induces a defense response in plants, the expression of key genes in defense-related pathways in tobacco was examined after DcPDI1 injection. These included genes related to the salicylic acid (SA) pathway (NPR1, PAL, PR1a) and genes related to the aasmonic acid (JA) pathway (PR3, PR4, LOX2, MYC2, JIP21).

[0047] Two days after injection of empty vector (EV) and Agrobacterium DcPDI1, tobacco leaves were harvested, and RNA was extracted and reverse transcribed. RNA extraction and reverse transcription were performed as in step 3. The qPCR system was as follows: 10 μL of 2 × SupRealQ Ultra HunterSYBR qPCR Master Mix (U+), 0.4 μL each of forward and reverse primers, 1 μL of cDNA template, and 8.2 μL of ddH2O. The reaction system was as follows: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 10 sec, extension for 60 sec, and 10 sec, for 40 cycles; finally, the instrument's default melting curve was added. The detection primer sequences were DcPDI1-qF: CTGAATTGGCCAAGACTTTC and DcPDI1-qR: CGGACCTTCGTAGTTGACGC. Internal reference primer sequences: NbRPL23-qF: AAGGATGCCGTGAAGAAGATGT, NbRPL23-qR: GCATCGTAGTCAGGAGTCAACC.

[0048] The expression of key genes in defense-related pathways in tobacco was detected 24 h after DcPDI1 injection. The results showed that the expression of genes related to the salicylic acid pathway was significantly inhibited. Figure 4 (a) of the study, while the expression of genes related to the jasmonic acid pathway was significantly increased. Figure 4 (b) in the middle.

[0049] 8. Expression of effector protein DcPDI1 in citrus psyllids Citrus psyllids of different instars (eggs, nymphs, and adults) were collected, and different tissues (head, thorax, abdomen, midgut, and antennae) were dissected for RNA extraction, reverse transcription, and qPCR. RNA extraction and reverse transcription were performed as in step 3. The qPCR procedure was the same as in step 7, with the detection primer sequences being DcPDI1-qF: CTGAATTGGCCAAGACTTTC and DcPDI1-qR: CGGACCTTCGTAGTTGACGC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, with the primer sequences being DcGAPDH-qF: CATGGCAAGTTCAACGGTGA and DcGAPDH-qR: CGATGCCTTCTCAATGGTGG.

[0050] The expression of DcPDI1 in different instars (eggs, nymphs, and adults) and different tissues (head, thorax, abdomen, midgut, antennae, and legs) of the citrus psyllid was detected by qPCR. Figure 5 and Figure 6 The results showed that the expression level of DcPDI1 varied across different larval stages and tissues. The expression level was significantly higher in the egg stage than in the nymph and adult stages; it was also relatively high in the midgut and abdomen, and significantly higher than in other tissues.

[0051] Adult citrus psyllids were collected before feeding (0 h) and at 12 h, 24 h, 36 h, and 48 h after feeding. RNA was extracted, reverse transcribed, and qPCR was performed. The RNA extraction and reverse transcription steps were the same as in step 3. The qPCR steps were the same as in step 7, and the primer sequences were the same as above (DcPDI1-qF, DcPDI1-qR, DcGAPDH-qF, DcGAPDH-qR).

[0052] The expression of DcPDI1 after consuming sweet oranges was examined. It was found that the expression level of DcPDI1 increased significantly with increasing consuming time, reaching a peak at 36 h. Figure 7 ).

[0053] 9. RNAi of DcPDI1 Following the method of Yu et al. (Yu X, Gowda S, Killiny N, 2017. Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri. Pestmanagement science, 73: 1846-1853), RNAi experiments on citrus psyllids were conducted using a feeding method. The main steps are as follows: First, dsRNA was synthesized using the T7 RiboMAX™ Express RNAi System kit (Promega, catalog number P1700) according to the manufacturer's instructions. Then, the dsRNA was diluted to 60 ng / μL. After starving adult citrus psyllids for 4 h, they were anesthetized with CO2 and 0.2 μL of the dsRNA solution was dropped onto the thorax and ventral side of the citrus psyllid between its three pairs of legs using a microsyringe. 60 ng / μL of dsRNA-GFP was used as a control. After 60 seconds of incubation for complete absorption, the samples were transferred to clean disposable petri dishes and then placed in an incubator at 26 ± 2 ℃, 60 ± 5% humidity, and a photoperiod of L:D = 16:8. Surviving adult citrus psyllids were collected at 12 h, 24 h, 36 h, 48 h, and 72 h to detect DcPDI1 expression, with five replicates. The oviposition rate of surviving female citrus psyllids after 72 h of dsRNA treatment was recorded within 24 h, with ten replicates. The developmental time of citrus psyllids after RNAi was analyzed, with a total of 30 individuals counted.

[0054] The primers for dsRNA are as follows: dsRNA-DcPDI1-F: TAATACGACTCACTATAGGG CTGACAGCAAGGTTGTTGTTG; dsRNA-DcPDI1-R: TAATACGACTCACTATAGGG GAGAAGAACACCAACAGATG; dsRNA-GFP-F: TAATACGACTCACTATAGGG GTCCTCGATGTTGTGGCGGA; dsRNA-GFP-R: TAATACGACTCACTATAGGG ACCACATGAAGCAGCACGAC; The underlined sequence is the T7 promoter sequence. RNAi studies investigated the function of DcPDI1 in the citrus psyllid, and the results showed that knockdown of DcPDI1 significantly reduced oviposition and developmental duration in the citrus psyllid. Figure 8 This indicates that DcPDI1 plays an important role in the growth and development of citrus psyllids.

[0055] The above findings suggest that DcPDI1 may be a novel molecular target that plays a key role in activating the host plant's defense response and assisting the citrus psyllid in feeding, providing new insights into the molecular mechanisms of the interaction between the citrus psyllid and the plant.

[0056] SEQ ID NO.1 (Amino acid sequence of DcPDI1) MRRLSLLFLLISPLVVFA DDVTEEDGVLVLTQDNFQSSIEKHDHILVEFYAPWCGHCKQLVPEYSKAALQLATDGHDIKLAKVDATQHTALAEQYGVRGYPTLKFFKKRSIIEYGGGRTAEDIVNWLLKKTGPPAKE FTSVDEIKAFIADSKVVVAGLFKDASSELAKTFNEIASKVDDLVFVTSTNADILAEYSVDDDTVAIFKKFDEGRVNYEGPASDEAALRKFLSTQSLPLVVEFNHETAQKIFGGEIKSHLL VFFSKAAGHYESHFEPVQTVAKDFREKVLFVTINTDEEDHQKILEFFGMSKDEVPSLLRLIRLEEDMAKYKPATSEISVDTVRSFVTEFLAGNLKQHLLSQPLPEDWDKNAVKVLVASNFD EIAFDKSKHVLVEFYAPWCGHCKQLAPIYDKLGEKFADRDDITIAKIDATVNELEHTKITSFPTLKLYAKDDNRVIDYNGERVLEALSNFVESGGKEGGLPSGAQEEQDEDDDQPKRDEL The underlined part represents the signal peptide sequence.

[0057] SEQ ID NO.2 ( DcPDI1 (nucleotide sequence) ATGCGCCGGTTATCCTTGTT CAAAGAGAGATGAGTTGTAA 。

Claims

1. A citrus psyllid effector protein, DcPDI1, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the citrus psyllid effector protein DcPDI1 as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. The application of the citrus psyllid effector protein DcPDI1 as described in claim 1 or the gene as described in claim 2 in the control of citrus psyllids.

4. The application according to claim 3, characterized in that, This refers to the application of a preparation that silences the gene of the citrus psyllid effector protein DcPDI1 in the control of citrus psyllids.

5. The application according to claim 4, characterized in that, The formulation is the dsRNA of the gene encoding the citrus psyllid effector protein DcPDI1.

6. The application according to claim 3 or 4, characterized in that, The control of citrus psyllids aims to reduce their egg production and development period.

7. The use of an agent that inhibits the citrus psyllid effector protein DcPDI1 as described in claim 1 or the gene as described in claim 2 in the preparation of a product for the control of citrus psyllids.

8. Inhibiting the use of the citrus psyllid effector protein DcPDI1 as described in claim 1 in regulating plant defense responses, wherein the plant is tobacco.

9. The application according to claim 8, characterized in that, The application is achieved by inhibiting the promotion of plant ROS by the citrus psyllid effector protein DcPDI1, inhibiting plant salicylic acid pathway-related genes, and / or promoting jasmonic acid pathway-related genes.

10. The application according to claim 9, characterized in that, The salicylic acid pathway-related genes are NPR1, PAL, and PR1a, and the jasmonic acid pathway-related genes are PR3, PR4, LOX2, MYC2, and JIP21.

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