An isofemale population of B. tabaci Asia II 1 being maintained at the whitefly rearing facility, Advanced Centre for Plant Virology, Indian Agricultural Research Institute (IARI), New Delhi since 2015 was used in the present study. The iso-female line was reared on eggplants, Solanum melongena (var. Navkiran, Mahyco, India) at 28 ± 2°C temperature, 60 ± 10% RH, and 16 hr light - 8 hr dark photoperiod. The identity of the pure culture was confirmed by sequencing of mitochondrial cytochrome oxidase subunit I (mtCOI).

The initial inoculum was taken from a pure culture of ChiLCV maintained at the laboratory by B. tabaci-inoculation. The culture was maintained on chilli plants (var. Priti, Nunhems) in insect-proof conditions. The identity of the virus was further confirmed by sequencing the DNA-A component amplified in PCR using primer pair, Begomo F-Begomo R (Akhter et al., 2009).

In our previous study, the expression of B. tabaci TLR3 and TOB1 was found highly abundant in response to ChiLCV infection (Nekkanti et al., 2022). In the present study, dsRNAs were designed to knock down B. tabaci TLR3 and TOB1. The conserved regions were identified by aligning the sequences of B. tabaci TLR3 and TOB1 available in NCBI. Putative siRNAs in the conserved regions were identified using the siRNA Wizard online tool (https://www.invivogen.com/sirna-wizard, accessed on 12-12-2021). The regions with the maximum number of siRNAs were selected for designing dsRNA. The dsRNA stretch was further investigated for off-target effects with other organisms like humans, mice, birds, ants, and bees. A dsRNA targeting Thrips palmi collagen alpha-1(III) chain-like (TpCOL3A1) and not specific to B. tabaci was used as negative control.

The primer pairs were designed using the NCBI primer blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) to amplify the dsRNA stretches. The primers were validated and PCR conditions were optimized in a gradient PCR. The primer pairs used in the study are listed in Table 1 . A 25 µL PCR mixture contained 1X PCR buffer (Thermo Fisher Scientific, USA), 0.4 µM of each forward and reverse primer (GCC Biotech, India), 0.26 mM dNTP mix (Thermo Fisher Scientific), 50 ng DNA template of B. tabaci, and 2 U of DreamTaq DNA polymerase (Thermo Fisher Scientific). PCR was performed in a T100 thermocycler (Bio-Rad, USA) with initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 40 s, annealing at 55°C for 40 s, extension at 72°C for 40 s, and a final extension at 72°C for 10 min. The amplified PCR products were resolved on 2% agarose gel along with 1 kb plus DNA ladder (Thermo Fisher Scientific) and visualized under a Gel documentation system (MaestroGen Inc, Taiwan).

The amplified PCR products were eluted, ligated in the L4440 expression vector between two T7 promoters, and sequenced for further confirmation. The recombinant plasmids were transformed into RNase III mutant E. coli HT115 cells. The recombinant E. coli HT115 cells were induced with 0.8 M isopropyl-β-D-1-thiogalactopyranoside (IPTG) and cultured overnight at 37°C in a shaking incubator. Total RNA from the induced HT115 cells was extracted using Trizol reagent (Invitrogen, CA, USA) and resuspended in nuclease-free water. The dsRNA was purified by incubating with 1 U of RNase A, DNase and protease-free (Thermo Fisher Scientific) and 1 U of DNase I, RNase-free (Thermo Fisher Scientific) for 1 hr at 37°C in the presence of 500 mM sodium chloride as described by Chakraborty and Ghosh (2022). The enzymes were inactivated by chloroform extraction. The purified dsRNA was quantified in a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific), and visualized on 2% native agarose gel stained with GoodView (BR Biochem, India).

The purified TLR3 and TOB1 dsRNAs were separately delivered to B. tabaci via the oral feeding method described by Chakraborty and Ghosh (2022). Briefly, around 30 flies were collected in each cylindrical pet bottle (3.5 cm diameter, 16 cm height). The open end of the bottle was sealed with a stretched UV-sterilized Parafilm M. Based on our previous study, purified dsRNA at 3.0 μg/mL was supplemented with the artificial diet comprised of 20% sucrose and 5% yeast extract. The diet with dsRNA was sandwiched between two layers of stretched Parafilm M membrane. A diet without dsRNA and diet with TpCOL3A1 dsRNA were served as control. For ventilation, a hole was made in the wall of the pet bottle and sealed with a muslin cloth. The pet bottles were kept in the upright position in dark at 26 ± 2°C and 60% RH. Three replicates were maintained for each treatment and repeated nine times. Percent mortality data was recorded 48 hr post dsRNA exposure. Tukey’s test was used to differentiate the mean differences across the categories with a 95% confidence interval using XLSTAT 2014.5.03. The surviving B. tabaci from these replicates were utilized to examine the relative expression of TLR3 and TOB1 mRNA and ChiLCV acquisition and transmission efficiency as described below.

The relative expression of B. tabaci TLR3 and TOB1 was estimated 48 hr post dsRNA feeding considering the β-actin gene as endogenous control. The primer pairs, AG301F-AG302R and AG568F-AG569R for TOB1 and TLR3, respectively were used in the RT-qPCR assay ( Table 1 ). Around 30 surviving B. tabaci in three replicates post TLR3 and TOB1 dsRNA exposure were used for total RNA isolation using Trizol reagent. The RNA was quantified in a spectrophotometer (NanoDrop 2000) and used for complementary DNA (cDNA) synthesis using the FIREScript RT cDNA synthesis kit (Solis BioDyne, Estonia). The 20 μL reaction mixture comprised of 1.0 μg RNA template, 5 μM oligo dT primers, 500 μM dNTP mix, 2 μL of 1 X reaction buffer, 10 U FIREScript RT, and 1 U RiboGrip RNase inhibitor. cDNA synthesis was carried out in a T100 thermocycler with reverse transcription at 42°C for 60 min, and enzyme inactivation at 85°C for 5 min. The relative RT-qPCR assay was performed in an Insta Q48M real-time PCR (Himedia, India). A 20 µL reaction mixture contained 1X GoTaq qPCR master mix (Promega, USA), 300 nM CXR passive reference dye, 0.25 µM of each forward and reverse primer, and 2 µL of template cDNA. Thermal cycling was performed at initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 40 s, 55°C for 40 s, and 72°C for 40 s. After each reaction, a dissociation or melting curve was performed to evaluate the specificity of the amplicons. Three biological and two technical replicates were used in the RT-qPCR. The relative expression of mRNA in dsRNA-fed B. tabaci was measured in comparison to untreated control following the 2 − Δ Δ CT method (Livak and Schmittgen, 2001). Microsoft Excel version 2016 was used to perform statistical analysis and prepare graphs. Expression of B. tabaci TLR3 and TOB1 in TpCOL3A1 dsRNA-fed flies was considered as the negative control.

A portion of surviving flies post-dsRNA exposure was used to quantify the virus titer in B. tabaci. The flies were allowed to acquire ChiLCV by feeding on a ChiLCV-infected chilli plant (var. Preeti) for 24 hr. The ChiLCV copies acquired by dsRNA-exposed and nonexposed B. tabaci were estimated by absolute quantification in qPCR. DNA was isolated from the batch of 30 adult flies in three replicates using a CTAB extraction buffer as described by Roy et al. (2021) and quantified in a spectrophotometer. qPCR was performed in Insta Q48M real-time PCR with ChiLCV-specific primer pair, AG149F-AG150R ( Table 1 ) (Roy et al., 2021). This was followed by a melting curve analysis to check the specificity of the reaction. Each treatment had three biological and two technical replicates. A standard curve of ChiLCV using primer pair AG149F-AG150R generated in our previous study (Chakraborty and Ghosh, 2022) was used to quantify the ChiLCV copies. The mean CT values obtained in qPCR were fitted into the standard curve and the resulting concentration was used for the calculation of virus copy number in Microsoft Excel 2016 using the following formula.

where N = number of viral copies, x = amount of amplicon in ng, and n = length of linearized plasmid DNA. The mean differences in virus copies were assessed for statistical significance by Tukey’s test at a confidence interval of 95% using XLSTAT 2014.5.03.

To check the transmission efficacy of dsRNA-treated B. tabaci, a portion of B. tabaci exposed to ChiLCV for 24 hr was released onto the healthy chilli plants (var. Preeti) at the 3-4 leaf stage. They were allowed for 24 hr of inoculation feeding and eliminated manually. Ten plants in three replicates were used and four adult females per plant were released. The plants were maintained under insect-proof conditions and monitored for symptom development. B. tabaci, not exposed to dsRNA, were used as control. The ChiLCV infection in the inoculated plants was confirmed by ChiLCV-specific PCR at 21 days post-inoculation (dpi).

The identity of the B. tabaci population was confirmed by the nucleotide sequence of mtCOI gene. PCR with primer pair C1-J-2195 and L2-N-3014 amplified ~600 bp product as visualized on 1% agarose gel. The sequence analysis with BLASTn showed 100% homology to B. tabaci Asia II 1. The sequence submitted to GenBank can be retrieved by Accession No. OP223446.

PCR amplified a 2.7 kb product of full-length DNA-A segment from ChiLCV-infected plants. The sequence of DNA-A showed 100% homology to ChiLCV isolates upon BLASTn analysis. The sequence can be retrieved by Accession No. OM513903.

Based on the multiple alignments of B. tabaci TLR3 and TOB1 sequences available in NCBI, the conserved 340 nt and 167 nt stretches of TLR3 (~5.6 kb) and TOB1 (~2.28 kb), respectively were chosen for dsRNA designing. The dsRNA sequences were unique to B. tabaci and no off-target hits were detected with Homo sapiens (taxid: 9605), Formicidae (taxid: 36668), mice (taxid: 10088), honeybees (taxid: 7460), and Aves (taxid: 8782) in blastn analysis.

PCR with primer pairs AG568F-AG569R and AG301F-AG302R produced amplicons of 340 bp and 167 bp for B. tabaci TLR3 and TOB1, respectively ( Supplementary Figure 1 ). The nucleotide sequences of the amplified products showed 100% homology with already available B. tabaci TLR3 and TOB1 sequences. The sequences can be retrieved by Accession No. OP784422 and OP219521. The dsRNA purified from total RNA using DNase I and RNase A produced single specific bands of ~340 bp and ~167 bp, respectively on 2% agarose gel ( Figure 1 ). The concentration of TLR3 dsRNA was 970.0 ng/µL, whereas it was 779.9 ng/µL in the case of TOB1 dsRNA.

The feeding of TLR3 and TOB1 dsRNAs significantly reduced the target gene expression in B. tabaci adults. In RT-qPCR analysis, the log 2 − Δ Δ CT value of B. tabaci TLR3 expression was 11.55 under normal conditions. Exposure to TLR3 dsRNA significantly declined the TLR3 mRNA level by 6.77-fold compared to the untreated control at 48 hr. The downregulation of target gene expression was consistent in all the biological replicates. Similarly, TOB1 dsRNA significantly down-regulated the TOB1 mRNA level by 3.01-fold ( Figure 2 ). The log2^−ΔΔCT value of B. tabaci TOB1 expression was 3.19 under normal conditions. The reduction in the target mRNA expression level of B. tabaci was significantly higher in TLR3 dsRNA exposure than TOB1 dsRNA at 48 hr after oral delivery. The expression of B. tabaci TLR3 and TOB1 post TpCOL3A1 dsRNA exposure was statistically at par with untreated control. There was no significant regulation of the endogenous control gene, β-actin between dsRNA-exposed and non-exposed B. tabaci populations which indicated the specificity of the TLR3 and TOB1 dsRNAs on the target mRNAs. The melting curve analysis in RT-qPCR showed that the primer pairs for TLR3, TOB1 and β-actin did not produce any secondary peaks that indicated the specificity of the reactions.

Feeding on TLR3 and TOB1 dsRNAs significantly altered the fitness of B. tabaci under controlled conditions ( Figure 2 ). TLR3 and TOB1 dsRNA feeding at a concentration of 3.0 μg/mL induced mortality in B. tabaci adults. A mortality of 30.66% was recorded in TLR3 dsRNA-fed B. tabaci 24 hr post-feeding, whereas it was 29.99% when fed on TOB1 dsRNA. The mortality further increased with an increase in the exposure period. Up to 47.32% mortality was recorded 48 hr post-feeding on TLR3 dsRNA. In the case of TOB1 dsRNA, the mortality increased up to 43.99% compared to B. tabaci fed on a diet without dsRNA (9.33%). However, no morphological deformities were recorded in the TLR3 or TOB1 dsRNA-fed B. tabaci when observed under a microscope ( Figure 2 ). Mortality induced by TLR3 and TOB1 dsRNAs was found to be significant at p<0.0001 at a confidence limit of 95%. There was no significant mortality of B. tabaci post TpCOL3A1 dsRNA exposure compared to untreated control.

Silencing of B. tabaci TLR3 and TOB1 significantly decreased the ChiLCV titer within B. tabaci. The mean ChiLCV copy number was 2.84 x 10⁷ in B. tabaci fed on the diet without dsRNA ( Figure 3 ). Exposure to TLR3 dsRNA at 3 μg/mL induced 45.58 -fold reduction (6.23 x 10⁵ copies) in the mean ChiLCV copy. The decrease in ChiLCV copy in TOB1 dsRNA-treated B. tabaci was comparatively lower than in TLR3 dsRNA treatment. The ChiLCV titer in B. tabaci was reduced by 10.75-fold (2.64 x 10⁶ copies) post TOB1 dsRNA exposure. There was no significant change in ChiLCV titer post TpCOL3A1 dsRNA exposure compared to untreated control.

B. tabaci that were not exposed to TLR3 and TOB1 dsRNAs successfully transmitted ChiLCV to the inoculated plants. 93.33% of the inoculated plants tested positive in PCR with ChiLCV-specific primers. The infected plants showed characteristic ChiLCV symptoms like vein clearing, curling, and twisting of leaves, reduction of leaf size, puckering, reduction in inter-nodal length, thickening of leaves, swelling of veins, and overcrowding of leaves ( Figure 3 ). Chilli plants inoculated by B. tabaci exposed to TLR3 and TOB1 dsRNAs showed no symptoms up to 21 dpi. No amplification specific to ChiLCV was recorded in PCR for the plants inoculated by dsRNA-fed B. tabaci.