1. Plant materials and TSWV inoculum
Datura stramonium plants were kept in a growth chamber at 25°C with a 16-h light/8-h dark photoperiod.
TSWV was a gift from the Nanjing Agricultural University. This virus was maintained on Datura stramonium plants (Wan et al. 2018). TWSV-infected plant tissue for the acquisition access period (AAP) was obtained by mechanical inoculation of 3-week-old D. stramonium plants or through F. occidentalis insect vectors (Zhao et al. 2016). Infected tissue was ground in a chilled mortar and pestle in 10 mL of general extract buffer (Agdia, Indiana, USA). Plants to be inoculated were dusted with carborundum and gently rubbed with a cotton swab wetted with inoculum. Twelve days after mechanical inoculation, the leaves appeared deformed, curled, chlorotic, stained and other symptoms.
2. Insect culture
Frankliniella occidentalis, a susceptible laboratory strain, was a gift from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. The F. occidentalis colony was maintained on green beans (Phaseolus vulgaris) at 25°C, 50% relative humidity, and an L16:D8 photoperiod (Montero-Astúa, Ullman, and Whitfield, 2016).
Fresh beans were placed in the insect cage for the adult thrips to lay eggs. After 3 days, the thrips were removed with a brush, and the beans were placed in a new cage. The larvae were allowed to incubate and were fed with D. stramonium with or without TSWV for 72 h and then fed with green beans until they emerged as adults. First instar larvae (L1s), second instar larvae (L2s), pupae, female adults, and male adults of thrips were collected according to the methods of previous studies (Akoth, Pascal, Hans-Michael, and Pappu, 2016; Zhi, LI, and Gai, 2010). Subsequently, different developmental stages were collected for total RNA extraction. The experiments were repeated six times for each developmental stage (n ≥ 6).
3. Cloning of the UBR7 gene
3.1. RNA isolation and first-strand cDNA synthesis
Total RNA of adult F. occidentalis with TSWV was extracted using TransZol Up Reagent (TransGen, Beijing, China). Then, RNA integrity was further afﬁrmed using agarose gel electrophoresis. Genomic DNA elimination and reverse transcription was conducted using TransScript® II One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Beijing, China). The synthesized cDNA was stored at −20°C for further use.
3.2. Cloning of the full-length UBR7 cDNAs
On the basis of the National Center for Biotechnology Information (NCBI) data (Schneweis, Whitfield, and Rotenberg, 2017), primer sequences were designed using Primer Premier 5.0 software to verify the full-length UBR7 gene (Table. 1). Then, according to the 2 × TransTaq® High Fidelity PCR SuperMix I (-dye) kit (TransGen, Beijing, China), PCR amplification was conducted in an Applied Biosystems Veriti™ Dx 96-Well Fast Thermal Cycler (Thermo, Massachusetts, USA). Then purified PCR products were ligated into the pClone007 Vector (Tsingke, Beijing, China) and were transformed into Trans5α Chemically Competent Cell (TransGen, Beijing, China). Positive clones were selected by ampicillin resistance and then sequenced by Tsingke Biotechnology in Beijing, China.
3.3. Gene characterization and phylogenetic analysis
UBR7 gene sequence analysis was performed using the NCBI Basic Local Alignment Search Tool (BLAST) program. ScanProsite was used to speculate functional sites. The molecular weight and isoelectric point of the deduced protein sequences were computed using the ExPASy Proteomics Server. The physicochemical properties of UBR7 were evaluated through Protparam. To analyze the sequence homology and phylogenetic relationships of UBR7, E3 ubiquitin-protein ligase gene information from different species (including Thrips palmi, Cryptotermes secundus, Pediculus humanus corporis, Nilaparvata lugens, and Laodelphax striatellus) was downloaded from GenBank. Then, a phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates through MEGA5 (Nei et al. 2013).
The amino acid sequences (AAs) were downloaded from NCBI GenBank (including Solanum, Nicotiana, Datura) for the multiple sequence alignment. To visualize the conserved motifs, the AAs were analyzed with Clustal and T-coffee. WebLogo illustrated the amino acid frequencies.
4. UBR7 gene interference
4.1. dsRNA synthesis
Primers for RNA interference (RNAi) was designed using Harvard's SnapDragon program based on UBR7 gene sequence. The T7 promoter sequence (-TAATACGACTCACTATAGGG-) was added to the 5′ of the forward and reverse primers. After pretest screening, we selected the dsRNA primer with the highest interference efficiency (Table. 1).
The PCR was then carried out using ds-UBR7 primers and the negative control EGFP (ds-EGFP) primers by referring to previous studies (Table. 1) (Xiang, Li, Guo, Jiang, and Huang, 2011). The T7 RiboMAXTM Express RNAi System (Promega, Wisconsin, USA) was used to synthesize dsRNA. After being measured the concentration, the samples were stored at −80°C.
4.2. dsRNA feed preparation
Sucrose was weighed and dissolved in DEPC water to prepare a solution with 30% mass fraction as feed. Then, the sucrose solution was used to dilute the dsRNA to the concentration of 500 ng/μL. There groups of feeds were subsequently prepared (blank control, CK: 30% sucrose + ddH2O; negative control group, ds-EGFP: 30% sucrose + dsRNA-EGFP; and experimental group, ds-UBR7: 30% sucrose + dsRNA-UBR7).
4.3. Interference efficiency and survival rate detection
Fifty adult thrips without TSWV were randomly selected regardless of gender. These thrips were placed into a separate plastic cup. They were fed after incubated for a 4 hour-starvation. After 24 h, the UBR7 expression level and the survival rate of were determined.
5. Experiments of TSWV acquiring
Collected trips in L1s were divided into three groups (CK, ds-EGFP, and ds-UBR7) and were fed an artificial diet for an AAP of 24 h. After AAP, thrips were transferred into cups, and were fed with D. stramonium plants carrying TSWV. After 48 h, the leaves were replaced with healthy beans, until the thrips reached to L2s. The TSWV abundance of L2 thrips was detected using real-time quantitative PCR (qPCR). The process workflow was shown in Figure 1(a).
6. Experiments of TSWV transmitting
Collected adult thrips with TSWV 3 days after eclosion and were divided into three groups (CK, ds-EGFP, and ds-UBR7), which were fed an artificial diet for an AAP of 24 h. After the AAP, the artificial diet was removed from the tube and replaced with the true leaf of a healthy D. stramonium, a small disc with a diameter of 5 mm. After 48 h, small disks were removed and placed in a 48-well plate, 1 mL ddH2O was added to each well, and the plates were placed in an illumination incubator for 72 h. A double-antibody sandwich enzyme-linked immunoassay method then was used to detect the TSWV infection rate of the leaves. The process workflow was shown in Figure 1(b).
7. Real-time qPCR
The expression levels UBR7 were examined using qPCR. Primer sequences were designed using Primer Premier 5.0 software or a reference research report (Table. 1) (Piao, Yao, He, and Fan, 2008; Qingjun et al. 2014). Then, according to the 2 × T5 Fast qPCR Mix (SYBR Green I) kit (Tsingke, Beijing, China), qPCR amplification (n = 6) was conducted in PCR system using a 20 μL reaction volume containing 10 μL 2 × T5 Fast qPCR Mix (SYBR Green I), 0.8 μL of primers (10 μM) and ~50 ng of cDNA. The reaction conditions were as follows: initial denaturation (1 min at 95°C) followed by 45 amplification cycles of 95°C for 10 s and 58°C for 60 s, and the fluorescence signal value was obtained at 60°C for 1 min. Melt curves were generated to confirm that only one specific PCR product was amplified and detected. The relative gene expression levels were calculated using 2−ΔΔCT. Actin was used as the reference gene.
8. Western blot analysis
Total protein was extracted from thrips at different developmental stages with a sodium dodecyl sulfate sample buffer and applied for western blotting using anti-β-actin (TransGen, Beijing, China) and anti-UBR7 antibodies (AtaGenix, Hubei, China). Anti-UBR7 was prepared by predicting the antigenic determinant through BepiPred-2.0, synthesizing the peptide (Peptide 1: CKRPYPDPEDTSDDE; Peptide 2: Cys+NTPGSSSQKSNIETP), and then preparing the antibody. The antibody-reactive bands were revealed using enhanced chemiluminescence (Beyotime, Shanghai, China) and detected using photographic film. The intensity of the bands was quantified using Quantity One.
9. Enzyme-linked immunosorbent assay
Then, 100 μL of PBS was used to grind and break the small disk in method 5 Experiments of TSWV acquiring. The samples were centrifuged at 10,000 × g for 5 min, and the supernatant was transferred to a new tube for TSWV Enzyme-linked immunosorbent assay (ELISA) (Mmbio, Jiangsu, China). The positive judgment criterion was that the absorbance values were greater than or equal to twice the negative control value (judgment criteria: positive value ≥ 2 × negative control value).
10. Surface plasmon resonance technology analysis
According to the bioinformatics analysis of UBR7, we know that it has two domains, zinc finger domain 51–115 AAs and PHD-SF superfamily 142–193 AAs, and 51–193 AAs was selected as the target fragment for expression. Using the full-length cloned plasmid as a template, the target fragment was subcloned into the pET28b+ vector. Escherichia. coli (E. coli) expressed the target protein, and a 6 × His tag was added to the N-terminal for expression and purification. The purified UBR7-domain protein and Nicotiana benthamiana (N. benthamiana) with TSWV were sent to AtaGenix for the SPR test. The protein eluate was analyzed with a LC-MS/MS, QE by BGI Genomics.
11. GST pull-down assay
The target fragment was subcloned into the pGEX-6P-1 vector using the full-length cloning plasmid of the TSWV N gene as a template. The target protein was expressed by E. coli, and the N-terminal GST tag was added for expression and purification.
The purified TSWV N-GST and GST proteins interacted with the UBR7 domino-His protein through BeyoMag™ Anti-GST Magnetic Beads (Beyotime, Shanghai, China). The pull-down results were analyzed using western blotting after incubation with anti-UBR7 and anti-GST (Beyotime, Shanghai, China).
12. Statistical analysis
The results were expressed as the mean ± standard error. In the following steps, all data were processed with SPSS version 19.0 (SPSS Statistics for Windows, Illinois, USA). Duncan's multiple range test followed by a one-way analysis of variance was used to compare the significant differences. Treatments not sharing a common letter were significantly different at P < 0.05. Alternatively, independent-sample t-tests were used to compare the differences between the two groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).