Small brown planthoppers

Viruliferous and non-viruliferous small brown planthopper strains used in this study were described previously. The viruliferous and non-viruliferous small brown planthopper strains used in this study were obtained from a field population collected at Hai’an, Jiangsu Province, China. The viruliferous strain contained the Jiangsu RSV isolate. Insect rearing was performed as described previously (Zhao et al., 2016a). Another RSV isolate, Yunnan isolate, was collected from Kunming in Yunnan Province and stored at −80°C (Zhao et al., 2018).

RNA isolation and cDNA synthesis

RNA was isolated from various planthopper samples and nuclear and cytoplasmic protein extracts using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. After being treated to remove genomic DNA contamination using a TURBO DNA-free kit (Ambion, Austin, TX, USA), 1 μg of RNA was reverse transcribed to cDNA using the Superscript III First-Strand Synthesis System (Invitrogen) and random primers (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions.

Sequence alignment and phylogenetic analysis

To identify importin α proteins and caspases in the small brown planthopper, human and D. melanogaster importin α proteins and caspases as well as N. lugens caspases were used as queries to search against the planthopper gene set (3) (Zhu et al., 2017) using BLASTp with an E-value cutoff of 1E-5. These sequences were aligned with ClustalW, and neighbor-joining phylogenetic trees were constructed with a bootstrap of 1000 using MegaX. Branches with <50% bootstrap value were collapsed.

Extraction of nuclear and cytoplasmic proteins and Western blot analysis

Nuclear and cytoplasmic proteins were extracted from viruliferous nymphs using a Nuclear and Cytoplasmic Extraction kit (Beyotime, Jiangsu, China). Thirty nymphs were homogenized in 200 μL of ice-cold cytoplasmic extraction A and B reagents (volume ratio of 20:1) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) using a TGrinder high-speed tissue grinder (Tiangen Biotech, Beijing, China). After incubating in ice bath for 15 min, the homogenate was centrifuged at 1,500 ×g for 5 min at 4 °C. The supernatant was retained as the first cytoplasmic protein fraction. Subsequently, another 200 μL of cytoplasmic extraction reagent A was added to the precipitate. After incubating in ice bath for 15 min, the homogenate was mixed with 10 μL of cytoplasmic extraction reagent B and centrifuged at 12,000 ×g for 5 min at 4 °C. The resulting supernatant was retained as the second cytoplasmic protein fraction. Both extracts were then mixed and used as cytoplasmic proteins. Subsequently, the precipitate was mixed with 50 μL of nuclear extraction reagent and vortexed for 30 min. After centrifugation at 12,000 ×g for 10 min at 4 °C, the supernatant was retained as the nuclear protein extract fraction. The cytoplasmic and nuclear protein extracts were used for Western blot analysis and RNA extraction. NP was detected using a homemade monoclonal anti-NP antibody. The reference proteins for nuclear and cytoplasmic proteins were histone H3 and GAPDH, which were detected using polyclonal anti-H3 antibody and anti-GAPDH antibody, respectively (Abcam, Cambridge, UK). The optical density of NP was quantified using Gelpro32 image analysis software and was normalized to that of histone H3. Differences were statistically evaluated in SPSS 17.0 using Student’s t-test.

Protein expression, purification and antibody preparation

The open reading frames (ORFs) of importin α1, α2, and α3 and fragments of importin α1 (352 to 528 aa), importin α2 (366 to 524 aa), and importin α3 (350 to 513 aa) were cloned and constructed into the vector pET28a between the restriction sites EcoRI and XhoI to generate His-tagged recombinant protein expression plasmids. The ORF for YY1 with a His-tag was constructed into pET28a between the restriction sites NdeI and EcoRI. The RSV ORF for NP with a Flag-tag was inserted into pET28a between the restriction sites NcoI and EcoRI. Four NP fragments [from 1 to 165 aa (N), 166 to 284 aa (C), 166 to 193 aa (C1), and 204 to 284 aa (C2)] with Flag-tags were inserted into pET28a between the restriction sites NdeI and EcoRI. The corresponding primers are listed in Table S3. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) for expression. After 10 h induction with 0.5 mmol/L isopropyl b-D-thiogalactoside (IPTG) at 16 °C, the cells were pelleted by centrifugation and then sonicated for 30 min on ice. Then, the supernatant from the sonicated cells was used for pull down or co-immunoprecipitation assays. The expressed fragments of importin α1, α2 and α3 were purified using Ni Sepharose (GE Healthcare, Buckinghamshire, UK) following the manufacturer’s instructions and served as antigens to produce rabbit anti-importin α1, α2, or α3 polyclonal antibodies by the Beijing Protein Innovation Company (Beijing, China).

Co-immunoprecipitation for Western blot analysis

For in vitro co-immunoprecipitation analysis, approximately 10% of the total recombinant proteins were used as input. To pull down the interacting proteins from the viruliferous planthoppers, total proteins were extracted from 4th-instar viruliferous planthoppers using 1× PBS buffer (pH 7.2) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Approximately 10% of the total protein fraction was reserved as input. Then, 10 μL of protein G beads (Thermo Fisher Scientific) was mixed with 2 μg of an anti-His/-Flag monoclonal antibody (Merck Millpore, Billerica, MA, USA) or anti-YY1 polyclonal antibody (Thermo Fisher Scientific) or a homemade anti-NP monoclonal antibody before being incubated with 200 μL of His- or Flag-tagged recombinant proteins for 15 min at room temperature. The expression products from the pET28a vector or the IgG antibody (Merck Millipore) were used as negative controls. Then, 500 μL of recombinant Flag- or His-tagged target proteins or the total proteins from viruliferous planthoppers were added and incubated at 4 °C overnight. Finally, the antibody-protein-protein complex was dissociated from the beads with the elution buffer (Thermo Fisher Scientific) for Western blot analysis.

Co-immunoprecipitation and mass spectrometry for identification of NP-interacting nuclear proteins

Protein G beads (10 μL) were mixed with 2 μg of a monoclonal anti-NP or IgG antibody (Merck Millipore) and then incubated with 500 μL of nuclear protein extracts from viruliferous planthoppers. Then, the antibody-protein-protein complex was dissociated from the beads with elution buffer (Thermo Fisher Scientific) for liquid chromatography-tandem mass spectrometry with a Q-Exactive instrument (Thermo Fisher Scientific) by the Beijing Protein Innovation Company. The mass spectrometry data were searched against the genome database of the small brown planthopper (Zhu et al., 2017). Peptides with scores higher than 80 were screened as putative NP-interacting nuclear proteins for further analysis.

Protein expression in S2 cells

The NP ORF was cloned into the plasmid pAc-5.1/V5-HisB (Invitrogen). The C2 fragment of NP (204 to 284 aa) and GFP were cloned into the plasmid pIEx-4 between the restriction sites KpnI and NotI to yield the plasmid pIEx-4-GFP-Gly-Ser (linker)-C2. The primers used for cloning are listed in Table S3, and the plasmids pAc-5.1/V5-HisB and pIEx-4-GFP were used as controls. For each plasmid, 20 ng was transformed into Drosophila S2 cells using Lipofectamine 3000 reagent (Thermo Fisher Scientific). The cells were sampled at 45 h after transfection and fixed with 4% (w/v) paraformaldehyde for 30 min. For NP-expressing cells, the monoclonal anti-NP antibody and Alexa Fluor 488 (green) affinipure goat anti-mouse IgG (YEASEN, Shanghai, China) were sequentially added. For GFP- and GFP-C2-expressing cells, GFP signals were directly observed. Nuclei were labeled with Hoechst (blue), and fluorescence was examined under a Leica TCS SP5 confocal microscope (Leica Microsystems, Solms, Germany).

Immunohistochemistry analysis

Midguts or salivary glands from viruliferous, non-viruliferous or dsRNA-treated planthoppers were dissected in 1× PBS buffer (pH 7.2) on a glass plate and fixed in 4% (w/v) paraformaldehyde for 2 h at room temperature. After being treated with osmotic buffer (0.01 mol/L phosphate-buffered saline containing 2% Triton X-100, pH 7.4) for 2 h, the tissues were incubated with the monoclonal anti-NP antibody and/or the polyclonal anti-importin α3 antibody overnight at 4 °C. After being washed with 0.01 mol/L phosphate-buffered saline containing 2% Tween-20 (pH 7.4), the tissues were blocked with 3% bovine serum albumin for 1 h. Then, the secondary antibody Alexa Fluor 488 (green) affinipure goat anti-mouse IgG or Alexa Fluor 594 (red) affinipure goat anti-rabbit IgG (YEASEN) was added. The nuclei were labeled with Hoechst (blue). The samples without the treatment of primary antibodies were used as negative controls. The images were viewed under a Leica TCS SP5 confocal microscope (Leica Microsystems, Solms, Germany).

Fluorescence in situ hybridization

The localization of RSV genomic RNAs in planthopper salivary glands and guts were performed using an RNA3 probe labeled with digoxigenin (DIG). The probe for RNA3 was synthetized using a T7/SP6 RNA Transcription kit (Roche, Basel, Switzerland) and was subsequently fragmented to approximately 250 bp via the alkaline lysis method. The primers used for RNA3 probe synthesis are listed in Table S3. Salivary glands and guts were dissected from viruliferous planthoppers and then fixed in 4% (w/v) paraformaldehyde at 4 °C overnight. After being digested with 20 μg/mL of proteinase K at 37 °C for 15 min, tissues were hybridized with 5 ng/μL of RNA3 probe at 37 °C overnight and then successively washed in 2×, 1×, and 0.2× SSC at 37 °C for 30 min twice. An anti-DIG alkaline phosphatase-conjugated antibody (1:500) was used for RNA3 probe detection. The DIG fluorescent signal was detected under an HNPP Fluorescent Detection Set (Roche). The samples without the treatment of RNA3 probe were used as negative controls. Images were viewed under a Leica TCS SP5 confocal microscope (Leica).

Colloidal gold immunoelectron microscopy

The midguts of viruliferous and nonviruliferous planthoppers were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at 4 °C overnight. After dehydration in 30%, 50%, 70%, 85%, 95% and 100% alcohol, the midguts were embedded in LR Gold Resin (Fluka Biochemika, Steinheim, Switzerland). Then, 70 nm-ultrathin sections of the embedded tissues were cut and placed onto 50-mesh copper grids before being blocked for 1 h in 100 mmol/L PBS containing 5% goat serum. The anti-NP monoclonal antibody (1:200) and 10 nm gold-conjugated goat-anti-mouse IgG (1:100) were sequentially added for 1.5 h incubations. Another group of sections from viruliferous insects was not treated with the anti-NP monoclonal antibody as a negative control. After being washed with PBS buffer five times, the grids were stained with 2% neutral uranyl acetate for 10 min in the dark. Then, the sections were viewed under a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV. Six sections of fifteen midguts from viruliferous or nonviruliferous insects were observed.

Double-stranded RNA synthesis and delivery

Double-stranded RNAs (dsRNAs) for importin α-1, 2 and 3, YY1, FAIM, and green fluorescent protein (GFP) were synthesized using the T7 RiboMAX Express RNAi System (Promega) following the manufacturer’s protocol. The corresponding PCR primers of dsRNA for these genes are shown in Table S3. A total volume of 23 nL of dsRNA at 6 μg/μL for each gene were delivered into the third-instar nymphs through microinjection using a Nanoliter 2000 instrument (World Precision Instruments, Sarasota, FL, USA). The corresponding primers of dsRNA for these genes are shown in Table S3.

Inoculation of small brown planthoppers with RSV crude preparations

RSV crude preparations were extracted from 50 viruliferous adult planthoppers with 100 μL of PBS buffer (pH 7.2) as previously described (Zhao et al., 2016a). Non-viruliferous fourth-instar nymphs were injected with 23 nL of a mixture of insect-derived RSV crude preparations and 6 μg/μL dsYY1-RNA using a Nanoliter 2000 instrument (World Precision Instruments). The control group was injected with a mixture of RSV crude extracts and dsGFP-RNA. After the injection, the planthoppers were raised on healthy rice seedlings.

Quantitative real-time PCR and reverse transcription-PCR

Quantitative real-time PCR (qPCR) was used to measure the relative RNA levels of NP and the genomic RNA3 of RSV and planthopper genes on a Light Cycler 480 II instrument (Roche). The primers for each gene are listed in Table S3. qPCR was performed in a volume of 10 μL comprising 1 μL of template cDNA, 5 μL of 2× SYBR Green PCR Master Mix (Fermentas, Waltham, MA, USA), and 0.25 μL of each primer (10 μmol/L). The thermal cycling conditions were 95 °C for 2 min followed by 45 cycles of 94 °C for 20 s, 60 °C for 30 s and 72 °C for 10 s. The PCR products were from 80 to 150 bp. Five to eight insects or 15 to 18 tissues per replicate and six to eight biological replicates were prepared. The transcript level of planthopper translation elongation factor 2 (EF2) was quantified to normalize the cDNA templates. The relative transcript level of each gene to that of EF2 was reported as the mean ± SE. Differences were statistically evaluated using Student’s t-test to compare the two means and one-way ANOVA followed by a Tukey’s test for multiple comparisons.

The four genomic RNAs of RSV were examined in the nuclear and cytoplasmic protein extracts by reverse transcription-PCR (RT-PCR) as described previously (Zhao et al., 2018) using the primers listed in Table S3. The PCR protocol was 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final incubation at 72 °C for 5 min.

Target prediction of YY1 and NLS prediction of NP

The binding sequences of the transcription factor YY1 were predicted in the promoter regions of the small brown planthopper genes (Zhu et al., 2017). Each promoter sequence was regarded as the 2,000-bp sequence upstream of the transcription start site of each gene. We predicted the potential targets of YY1 using the algorithms PROMO with the TRANSFAC 8.3 database (http://alggen.lsi.upc.es) based on the conservative YY1 binding motif (5′-CCGCCATNTT-3′) (Kim and Kim, 2009). Six candidate targets relative to the immune responses were verified via qPCR and ChIP-qPCR. The NLS of NP was predicted using cNLS mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) with a cutoff score of 2.

ChIP-qPCR analysis

ChIP was performed using a SimpleChIP plus Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA, USA) in accordance with the manufacturer’s instructions. One hundred fifth-instar nymphs from nonviruliferous planthoppers were crushed and treated with 1.5% formaldehyde for 15 min before being incubated with 125 mmol/L glycine for 5 min. Then, 1× buffer A containing 0.5 mmol/L DTT and 1× protease inhibitor cocktail (PIC) were added. The DNA was digested with 100 µL of micrococcal nuclease (1× buffer B, 0.5 mmol/L DTT, 1× PIC, and 0.5 µL of micrococcal nuclease) for 20 min at 37 °C and then incubated with 5 mmol/L EDTA for 10 min on ice. The pelleted nuclei were sonicated with three cycles of 20 s on and 30 s off. After centrifugation, the supernatant was diluted with 1× ChIP buffer (containing 1× PIC), and 10 µL of the diluted samples was set aside as the 2% input DNA. Immunoprecipitation was performed using anti-histone H3 (D2B12; Cell Signaling Technology), normal rabbit IgG (Cell Signaling Technology), or anti-YY1 polyclonal antibody (Thermo Fisher Scientific) together with ChIP-Grade Protein G Magnetic Beads at 4 °C overnight. After sequentially being washed with low salt buffer three times, high salt buffer three times, and TE buffer two times, DNA was eluted in 1× ChIP elution buffer for 30 min at 65 °C followed by addition of proteinase K and another 2 h incubation at 65 °C. The ChIP-enriched DNA was purified using spin columns following the manufacturer’s instructions before being used for qPCR analysis on a Light Cycler 480 II instrument (Roche). The promoter regions of six putative target genes of YY1 were amplified via qPCR using specific primers (Table S3). The promoter region of GAPDH1 was quantified in the anti-histone H3 immunoprecipitation as positive control. The thermal cycling conditions were as follows: 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The qPCR results were analyzed using the percent input method, the equation for which is shown below. Using this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin. ChIP-qPCR was repeated three times.

$$}\;} = \, 2\% \, \times \, 2^}\,2\% \;}\;}}\;}\;})}}$$

To compare the amount of FAIM promoter DNA that bound with YY1 from viruliferous and nonviruliferous planthoppers, ChIP was performed in viruliferous and nonviruliferous nymphs as described above. After quantifying the YY1 protein level using ELISA (Zhao et al., 2016a) and Western blot assay, the DNA contained in the comparable amounts of YY1 was extracted and the FAIM promoter was quantified with qPCR using specific primers (Table S3). IgG was used instead of the anti-YY1 antibody in the immunoprecipitation step as the negative control. Eight biological replicates were prepared for each group. The relative enrichment of FAIM promoter DNA binding with YY1 to that of IgG immunoprecipitation group (fold enrichment) were compared between viruliferous and nonviruliferous planthoppers and the difference was statistically evaluated using Student’s t-test.

Determination of caspase activity in planthoppers

Caspase activities were determined in viruliferous or nonviruliferous four-instar nymphs using human caspase 1, 3 and 8 Activity Assay kits (Beyotime Biotechnology) and a SpectraMax® Paradigm® Multi-Mode Detection Platform (Molecular Devices, San Jose, CA, USA) according to the manufacturer’s instructions.

Standard curves were constructed based on the molarities of p-nitroaniline (pNA) and OD405 values. The specific substrate was Ac-YVAD-pNA for caspase 1, Ac-DEVD-pNA for caspase 3, and Ac-IETD-pNA for caspase 8. Caspase activities were calculated as the product pNA (µmol/L). The slope and square of the correlation coefficient (R2) of each standard curve was calculated. The activities were measured at 7 d after the injection of dsRNAs. Twenty nymphs were used in one replicate, and four to six replicates were prepared for each group.

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

Midguts were dissected from three groups of planthoppers: viruliferous and nonviruliferous four-instar nymphs; nymphs 7 d after dsRNA-injection; and nymphs 6 d after the injection of 13.8 nL of 25 μmol/L pan-caspase inhibitor Z-VAD-FMK (Promega) or dimethyl sulfoxide (DMSO), performed in duplicate. After being fixed in 4% (w/v) paraformaldehyde and treated with the osmotic buffer, the midguts were used for TUNEL staining with a One Step TUNEL Apoptosis Assay kit (Beyotime Biotechnology) following manufacturer’s instructions. NP was assessed using the monoclonal anti-NP antibody in immunohistochemistry assays as described above. Fluorescence images were examined using a Leica TCS SP5 confocal microscope (Leica). The midguts treated with the pan-caspase inhibitor or DMSO were also sampled to quantify the RNA levels of NP and the genomic RNA3 of RSV via qPCR. Eight biological replicates and 15 to 18 midguts per replicate were prepared for qPCR.