(light:dark) photoperiod. A polymerase chain reaction (PCR) assay (Ko et al., 2007) was employed to examine the purity of the colony every three months.
The TYLCTHV- and ToLCTWV-infected tomato plants were provided by AVRDC—The World Vegetable Center and maintained in tomato plants cv. ANT22 by whitefly-mediated passage. All plants used in the experiments (tomato and Chinese kale) were grown from seeds under the same aforementioned condition. Tomato seedlings with three to five leaves were used as test plants for transmission assays.
2.2 Construction of expression plasmids of viral proteins
The open reading frames (ORFs) of TYLCTHV CP, TYLCTHV MP and Banana bunchy top virus (BBTV) CP (GenBank accession numbers: GU723742, GU723754,
and AY534140, respectively) were amplified from total DNA of virus-infected plants by PCR. DNA extraction and PCR were performed using DNeasy Blood & Tissue kit (Qiagen) and KOD plus DNA polymerase (Toyobo) following the manufacturers’
instructions. BBTV is transmitted by aphids in a persistent-circulative manner (Watanabe and Bressan, 2013), so BBTV CP was used as a negative control. The primer set for cloning TYLCTHV CP was 5’-GGGAATTCATGTCGAAGCGTCC
AGCAGA-3’ and 5’-GGCTCGAGATTCGTCACTGAGTCATAGAAA-3’. The primer set for cloning TYLCTHV MP was 5’-GGGAATTCATGGAGTCCAG AACTAACAATA-3’ and 5’-GGCTCGAGTATTTTCTTTGCATTAGAAGAGAC-3’.
The primer set for cloning BBTV CP was 5’-GGGAATTCATGGCTAGGTATCC GAAGAA-3’ and 5’-GGCTCGAGAACATGATATGTAATTCTGTTCTG-3’. EcoRI and XhoI recognition sites underlined were incorporated into the 5’ ends of the forward and reverse primers, respectively, to facilitate directional cloning. The PCR products were ligated into the pET32a vector (Novagen) and then transformed into E. coli strain BL21 (RBC Bioscience). pET32a vector was chosen because of its His tag and S tag sequence for purification and detection. Successful transformations were examined by restriction enzyme digestion and DNA sequencing.
2.3 Expression and purification of recombinant proteins
The transformed E. coli cells were cultured in LB broth (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) containing ampicillin (100 μg/mL) at 37°C until the OD600
reached 0.6-0.8. Protein expression was induced by 0.1 mM isopropyl beta-D-thiogalactopyranoside (IPTG). Affinity purification was used to purify the 6xHis-tagged recombinant proteins using Ni Sepharose High Performance (GE Healthcare). Bacterial cells were pelleted and lysed with binding buffer (20 mM sodium phosphate, 20 mM imidazole, 8 M urea, pH 7.4) by ultrasonication. The supernatant were collected after centrifugation (10000 g, 30 min) and applied to the column packed with Ni sepharose. The columns were then washed with binding buffer until the absorbance reached the baseline. The 6xHis-tagged proteins were then eluted with elution buffer (20 mM sodium phosphate, 500 mM imidazole, 8 M urea, pH 7.4). The
eluted proteins were re-purified using the same column and subsequently dialyzed against phosphate buffered saline (PBS) using Slide-A-Lyzer G2 Dialysis Cassette (Thermo Scientific) following the manufacturer’s instructions. Some precipitates occurred after dialysis, and soluble proteins in the supernatant were collected. The concentration of the recombinant proteins was quantified by Pierce 660 nm Protein Assay (Thermo Scientific) following the manufacturer’s instructions. The protein solutions were stored at -80°C for later usage.
2.4 SDS-PAGE
The purity of the recombinant proteins was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The eluted proteins were separated by electrophoresis in 10% polyacrylamide gels, and stained with Bio-Safe Coomassie (Bio-Rad) following the manufacturer’s instructions. Parafilm for 24 h. A green food dye (McCormick) was used to ensure that the whiteflies have ingested the medium. Four treatments were as followed: (1) TYLCTHV CP, (2) TYLCTHV MP, (3) BBTV CP, and (4) medium only. BBTV CP was included to test the specificity of viral proteins binding to the midgut cells of B. tabaci. The whiteflies were then dissected in PBS, and the midguts were fixed in 4% paraformaldehyde in PBS for
30 min. After fixation, the fixative was removed by washing three times with PBS. The samples were then permeabilized in PT buffer (PBS, 0.1% Triton X-100) for 30 min.
The midguts were subsequently incubated in blocking buffer (PBS, 1% BSA) for 1 h.
After which, the midguts were incubated in a mouse anti-S tag antiserum (Bioman) at a 1:200 dilution in PBT buffer (PBS, 0.1 % BSA, 0.1% Triton X-100) for 1.5 h. After washed three times with PT buffer, the samples were incubated in a goat anti-mouse IgG antiserum conjugated with Alexa fluor 633 (Molecular Probes) at a 1:200 dilution in PBT buffer for 1 h. The samples were washed again with PT buffer three times. The dissected midguts were mounted in SlowFade Gold antifade reagent with DAPI (Molecular Probes) and examined with a Leica TCS SP5 II confocal laser scanning microscope. Images were collected using the same laser power and gain. The in vivo binding assay was repeated three times, and three replicate insects were examined each time.
2.6 Competition for the virus binding site in midguts
Adult whiteflies were fed recombinant TYLCTHV CP, TYLCTHV MP, or BBTV CP in artificial feeding medium or medium only through membrane feeding for 24 h as
mixture of a goat anti-mouse IgG antiserum conjugated with Alexa fluor 633 (Molecular Probes) and a goat anti-rabbit IgG antiserum conjugated with Alexa fluor 555 (Molecular Probes) at a 1:200 dilution in PBT buffer for 1 h. The samples were washed again with PT buffer three times. The dissected midguts were mounted in SlowFade Gold antifade reagent with DAPI (Molecular Probes) and examined with a Leica TCS SP5 II laser scanning confocal microscope. Images were collected using the same laser power and gain. The experiments were repeated three times, and three replicate insects were examined each time.
2.7 Inhibition of TYLCTHV acquisition
To access if preacquisition of TYLCTHV CP by whiteflies would inhibit the acquisition of TYLCTHV, a virus quantification assay was employed. Adult whiteflies were fed recombinant TYLCTHV CP, TYLCTHV MP, or BBTV CP in artificial feeding medium or medium only through membrane feeding for 24 h as described above for the in vivo binding assay. The whiteflies were then transferred to TYLCTHV-infected
tomato plants to acquire the virus for another 24 h and subsequently fed a 15% sucrose solution for 6 h to clear the viruses remaining in the gut lumen. For each treatment, 20 adult whiteflies were collected, and their DNA was extracted using DNeasy Blood &
Tissue kit (Qiagen) following the manufacturer’s instructions. TYLCTHV in the tested whiteflies was quantified by real-time PCR. The primer set for TYLCTHV
quantification was 5’-CACGCCCGTCTCGAAAGT-3’ and
5’-GCTGTTGTATGGGCTGTCGAA-3’. The primer set for the quantification of endogenous Heat shock protein 90 (HSP90) gene was 5’-ATCGCCAAATCTGG AACTAAAGC-3’ and 5’-GTGTTTTGAGACGACTGTGACGGTG-3’ (Li et al., 2013).
The reaction was carried out using Fast SYBR Green Master Mix (Applied Biosystems) following the manufacturer’s instructions. The relative abundance of TYLCTHV DNA in the tested whiteflies was normalized to HSP90 gene. The experiments were repeated four times.
2.8 Inhibition of TYLCTHV transmission
To access if preacquisition of TYLCTHV CP by whiteflies would inhibit the whitefly transmission of TYLCTHV, a transmission experiment was employed. Adult whiteflies were fed recombinant TYLCTHV CP, TYLCTHV MP, or BBTV CP in artificial feeding medium or medium only through membrane feeding for 24 h as described above for the in vivo binding assay. The whiteflies were then transferred to TYLCTHV-infected
tomato plants to acquire the virus for another 24 h. After which, five whiteflies were used to inoculate each healthy test plant for a 24 h inoculation access period (IAP). The whiteflies were removed from the test plants after the IAP, and then a systemic insecticide (acetamiprid, diluted 1:1500) was used to kill all remaining whiteflies on the plants. Whitefly-inoculated plants were maintained in insect-proof Bugdorms (47.5 x 47.5 x 93 cm3) (Megaview) within a growth chamber under the same abovementioned conditions for disease development. A PCR assay (Weng et al., 2015) was employed to detect the virus infection in the test plants two weeks after inoculation. Ten replicate test plants were inoculated for each treatment, and the experiments were performed five times.
2.9 Inhibition of ToLCTWV acquisition and transmission
To determine if preacquisition of TYLCTHV CP by whiteflies would reduce the
virus binding to midgut cells, virus acquisition, and whitefly transmission of ToLCTWV, similar experiments were conducted as described above. To examine the competition between TYLCTHV CP and ToLCTWV for the virus binding site in the midgut cells of B. tabaci, adult whiteflies were fed recombinant TYLCTHV CP and ToLCTWV-infected tomato plants successively, and the whiteflies were dissected, fixed, and processed to immunofluorescence assays. The samples were treated with a mixture of a mouse anti-S tag antiserum (Bioman) and a rabbit anti-ToLCTWV antiserum at a 1:200 dilution. Primary antibody binding was detected with a mixture of a goat anti-mouse IgG conjugated with Alexa fluor 633 (Molecular Probes) and a goat anti-rabbit IgG conjugated with Alexa fluor 555 (Molecular Probes) at a 1:200 dilution.
The dissected midguts were examined with confocal microscopy. The experiments were repeated three times, and three replicate inscets were examined each time. ToLCTWV in the tested whiteflies was quantified by real-time PCR. The primer set for ToLCTWV
quantification was 5’-GCGACCCGCCGATATAGTC-3’ and
5’-CAGACGGCGACGAACCTT-3’. The relative abundance of ToLCTWV DNA in the tested whiteflies was normalized to HSP90 gene. The experiments were repeated three times.
To access if preacquisition of TYLCTHV CP would inhibit whitefly transmission of ToLCTWV, adult whiteflies were fed recombinant TYLCTHV CP and ToLCTWV-infected tomato plants successively, and five whiteflies were used to inoculate each healthy test plant. Disease development and PCR assay were conducted as described above. Ten replicate test plants were inoculated for each treatment, and the experiments were performed five times.
2.10 Statistics
Data obtained by real-time PCR were normalized and analyzed by one-way ANOVA, using the differences between the Ct value of TYLCTHV/ToLCTWV and HSP90 gene. Data from the transmission assays were analyzed by χ2 test.