B. tabaci B biotype (MEAM1) populations used in this study were reared on cotton seedlings (Gossypium hirsutum L. cv. pima) maintained inside insect-proof cages within growth chambers at 26°C, 60% relative humidity and a 14 h light/10 h dark photoperiod. One-week-old adult whiteflies were used for the feeding experiments. Tomato seedlings (Solanum lycopersicum cv. Avigail) plants were agroinoculated with partial tandem repeats (PTR) construct of TYLCV DNA A (Navot et al., 1991), and new infected plants were generated by whitefly-mediated inoculation which were used as the virus source for the experiments in this study. The purity of B. tabaci B biotype was confirmed using microsatellite Bem 23 primers and cytochrome C oxidase (COI) gene sequencing (De Barro et al., 2003). Presence of TYLCV in insects and plants was confirmed using the V1 and C473 primers (Ghanim et al., 1998) (Supplementary Table S1). Tomato plants at their three to five true leaf stages were used for virus transmission experiments.

Total RNA of B. tabaci and tomato plants were extracted using TriZol reagent (Invitrogen, United States) and the first strand cDNA was synthesized using Verso cDNA kit (Thermo scientific, Fermentas) following the manufacturer’s instructions. Fragments of B. tabaci cypB and hsp70 genes were amplified from the cDNA with Bt CypB F/R and Bt Hsp70 F/R primers pairs, respectively (Supplementary Table S1). For silencing tomato CypB gene as a control, a fragment of 302 base pairs was amplified using tomato cDNA as a template with the primers Tom CypB F/R primers pairs. All the fragments were T/A cloned into the pGMET vector (Promega), excised using EcoR1 and BamH1 and ligated to Tobacco rattle virus 2 (TRV 2) using the same restriction enzymes. The silencing TRV vectors including TRV1 and TRV2 were kindly provided by Prof. Henryk Czosnek from the Hebrew University of Jerusalem. The tomato gene Hsp90 was chosen as positive control to evaluate the silencing effect mediated by TRV, while TRV1 and TRV2 alone were used a negative control.

The TRV1 and four TRV2 constructs: TRV2-BtCypB, TRV2-BtHsp70, TRV2-TomCypB and TRV2-TomHsp90 were separately transformed into Agrobacterium tumefaciens C58 strain. The transformed A. tumefaciens cells were grown at 28°C in Luria–Bertani (LB) medium containing kanamycin and rifampicin (50 μg/mL each) for 24 h. pellets were resuspended in 10 mM MgCl₂, 10mM MES (2-(N-Morpholino) ethanesulfonic acid) and 120 mM acetosyringone) and kept at 25°C for 3 h. The suspensions of A. tumefaciens cells were diluted to OD₆₀₀ of 0.8. Each recombinant TRV2 virus construct was mixed with TRV1 virus in equal concentrations and inoculations of 2-week-old 30 tomato seedlings was performed by agro-infiltration (Senthil-Kumar and Mysore, 2014). Agroinoculated plants were maintained under 25 ± 2°C in growth rooms and phenotypic changes were recorded periodically. After 7 days post inoculation (dpi), the movement of TRV1 and four TRV2 constructs in tomato plants was detected by reverse transcription PCR (RT-PCR) analysis with vector and target gene specific primers (Supplementary Table S1). PCR positive plants were later transferred to insect-proof cages for insect bioassays.

B. tabaci RNAi bioassays were performed with 2-week-old plants after agroinoculation with the respective construct. Two plants from each combination, TRV1+TRV2-BtCypB, TRV1+TRV2-BtHsp70, and TRV1+TRV2 as control were transferred to individual insect-proof cages. For feeding experiments, newly hatched whiteflies from cotton plants were used as non-viruliferous insects. Other newly hatched whiteflies were transferred to TYLCV-infected tomato plants for 48 h acquisition access period and those were used as viruliferous insects. For each replicate around 300 viruliferous or non-viruliferous whiteflies were transferred to each group of plants in insect-proof cages. Quantification of target gene mRNAs was carried out from adults that were fed on the plants for 24 h, 72 h, and 168 h. After 1 week, additional insects were removed from the plants for analyzing the progeny. Plants were monitored every day for counting egg hatch and nymphal development. First generation progeny (nymphs) on all groups of TRV plants were collected and used for quantitative Real Time PCR (qRT-PCR), fluorescent in situ hybridization (FISH) assays and for light microscopy.

B. tabaci mortality assay was performed using 15 adult female whiteflies which were fed on all plant groups using clip-on-leaf cages. After 48 h, adults were removed from the leaf cage, and the exposed leaf area was marked with marker and eggs were counted. Plants were shifted to insect free cages and were monitored every 2 days for mortality and fecundity tests. Egg mortality was assessed after 8–10 days by counting number of viable nymphs. Eggs that had failed to hatch or dried out, or nymphs that had died on hatching, were scored as dead. Plants were maintained to collect first generated whiteflies. About 15 first generation female whiteflies were collected from the above assay and were fed on new tomato plants leaves to test fecundity. The experiments were repeated three times and five biological replicates were included in each experiment.

For total RNA extractions, adult whiteflies (30 whiteflies/ replicate) and nymphs (50 nymphs/replicate) were collected at each time point from tomato plants and RNA was isolated using TRIzol reagent (Sigma-Aldrich) then was treated with DNaseI according to the manufactures recommendations (Thermo Scientific). RNA (100 ng) was used as a template for cDNA synthesis in 25 μl reaction mixtures by using Verso cDNA kit (Thermo Scientific, Fermentas). For DNA isolation, a pool of 50 nymphs per replicate was isolated using CTAB (cetyltrimethylammonium bromide) method (Shahjahan et al., 1995). The same DNA was used for the screening all three symbionts Portiera, Hamiltonella, and Rickettsia using 16S rDNA specific primers (Supplementary Table S1). PCRs were carried out as previously described (Chiel et al., 2007). For qPCR, 3 ng of DNA was used in triplicates for each DNA sample. Five to six biological replicates and three technical ones for each biological replicates were used in all experiments.

The plant DNA was extracted from 100 mg of tomato leaf tissue, as described previously (Kanakala and Ghanim, 2016a). For qPCR, 50 ng of DNA was used in triplicates for each DNA sample. RNA was extracted from 100 mg of leaf tissue using TRIzol (Sigma-Aldrich), and the cDNA synthesis was performed as instructed by the manufacturer using Verso cDNA kit (Thermo Scientific, Fermentas). To ensure the validity of the data, cDNA was used for qRT-PCR in triplicate for each cDNA sample.

Target mRNAs and TYLCV in adult whiteflies and bacterial density in nymphs were quantified by qRT-PCR using Fast SYBR Green Rox mix (Thermo Scientific) using Rotor-Gene 6000 machine (Corbett Robotics Pty Ltd., Brisbane, QLD, Australia) and the accompanying software for qPCR data processing and analysis. The cycling conditions were: 15 min activation at 95°C, 45 cycles of 10 s at 95°C, 20 s at 60°C and 25 s at 72°C. Tomato β-actin and B. tabaci β-actin (Sinisterra et al., 2005), α-tubulin/BT-C02/BT-E06 (Mahadav et al., 2009) genes were used as internal controls for normalization after validation. The primers used for tomato and B. tabaci and symbionts are given in Supplementary Table S1. All assays were carried out in triplicates in each of three biologically independent experiments.

FISH was performed as previously described (Gottlieb et al., 2006). Briefly, nymphs were fixed overnight in Carnoy’s fixative (chloroform:ethanol:glacial acetic acid, 6:3:1, vol/vol). After fixation, the samples were decolorized in 6% H₂O₂ in ethanol for 2 h and then hybridized overnight in hybridization buffer (20 mM Tris–HCl pH 8.0, 0.9 M NaCl, 0.01% wt/vol sodium dodecyl sulfate, 30% vol/vol formamide) containing 10 pmol fluorescent probe/mL. For specific targeting of Portiera Rickettsia and Hamiltonella, the probes BTP1-Cy3 (5′-Cy3-TGTCAGTGTCAGCCCAGAAG-3′) (Gottlieb et al., 2006), Rb1-Cy5 (5′-Cy5-TCCACGTCGCCGTCTTGC-3′) (Gottlieb et al., 2006) and BTH-Cy5 (5′-CCAGATTCCCAGACTTTACTCA-3′) (Gottlieb et al., 2008), respectively, were used. Nymphs were stained with DAPI (4′,6′-diamidino-2-phenylindole) at 0.1 mg mL^-1. The stained samples were mounted whole in hybridization buffer and viewed under an IX81 Olympus FluoView500 confocal laser-scanning microscope. For each treatment, 20–30 nymphs were viewed. Optical confocal sections (100-μm thick) were sometimes prepared from each specimen for better visualization of the signal.

To assess the implication of CypB and Hsp70 in the transmission of TYLCV, 1-week-old B. tabaci adults were given acquisition on TRV-BtCypB and TRV-Bthsp70 inoculated plants for a week. The insects were then transferred to TYLCV-infected tomato plants for additional 48 h acquisition access period and subsequently single whiteflies were transferred to tomato plants in their three-leaf stage for 7 days. Whiteflies fed on TRV alone and TYLCV-infected plants for 48 h served as a controls. Tomato plants were grown in a potting mix in 1.5L pots under artificial light and maintained inside insect-free greenhouse under controlled temperature as detailed above. The whiteflies that were incubated with the plants were tested for TYLCV acquisition. The plants were monitored for the development of disease symptoms after 28 days post inoculation. DNA was extracted from symptomatic and non-symptomatic tomato plants and subjected to PCR for detecting TYLCV with specific coat protein (CP) primers V61 and V473 (Ghanim et al., 1998). The experiments were triplicated with 20 plants for each replicate.

A one-way analysis of variance using Tukey’s HSD adjustment test for multiple comparisons at a family-wise error rate of 5% was used for comparing all possible pairs of treatments. The connected letters in Figure 1–3, 5–7 provide a graphical summary: treatments not connected by the same letter are significantly different (i.e., Tukey adjusted p-value < 0.05). Error bars on the barplots are standard error of mean.

Tomato plants transiently expressing dsRNA of B. tabaci and tomato target genes were generated by agroinfiltration. After a week, total RNA from agroinfiltrated plants was subjected to reverse transcription-PCR to amplify the target genes and TRV using specific primers. Amplification of the desired bands and virus confirmed the systemic movement of the TRV and target gene mRNA required for inducing siRNA production. First, the working system was tested for the ability to silence tomato genes. Plants agroinfiltrated with pTV::Tom-CypB construct, made for silencing the tomato CypB gene caused significant leaf petiole rolling and leaf crinkling, while the plants remained retarded until 8 weeks after infiltration, and those symptoms extended until death (Figure 1A–C). The expression of the tomato CypB gene in those agroinfiltrated plants was monitored using qRT-PCR analysis, and the results showed significant decrease in the transcript levels at 7 dpi which were further reduced to significantly lower levels by 14 and 21 dpi (Figure 1G). The expression of the tomato CypB gene, however, remained stable in the control plants which were not infiltrated or which were infiltrated with control TRV1 and TRV2 constructs (Figure 1G). Tomato plants were also co-infiltrated with pTV::Tom-Hsp90 construct targeted to the endogenous heat shock protein 90 gene of tomato (Moshe et al., 2016) as an additional positive control. The virus propagation was visualized in stems 1 week after agroinfiltration and the stem became necrotic and plants died 24 days post infiltration as previously described (Figure 1D–F) (Moshe et al., 2016). The levels of the tomato Hsp90 transcripts over time showed gradual decrease over 21 dpi (Figure 1H), compared to the controls in which the levels of the transcript remained stable over the course of the experiment. Altogether, the above results confirmed the ability of the TRV system to knock down the expression of tomato target genes to very low levels and cause significant phenotypes in the plant that resulted in death.

Selected regions of the cDNA of CypB and Hsp70 genes were used for dsRNA expression in tomato plants (Supplementary Figure S1). The CypB gene codes for a Cyclophilin B gene (accession number KX268377) and the RNAi construct was designed to target the conversed two β sheets and α-helices from amino acids 1–77 which are conserved in B. tabaci compared to other arthropods (Kanakala and Ghanim, 2016a). This gene family exhibits a peptidylpropyl cis–trans isomerases activity (PPIases) and highly conserved Cyclosporin (CsA)-binding residues. On the other hand, the Hsp70 gene codes for heat shock protein 70, which are important for protein folding and responding to a variety of stresses in the cell (Morano, 2007). Hsp70-RNAi construct was designed to be specific to nucleotides unique to the conserved protein domain family NLPC_P60. B. tabaci CypB and Hsp70 are sufficiently different from the tomato CypB and Hsp70 genes, thus they were expected to be suitable for knockdown and obtaining specific results for B. tabaci genes. The CypB and Hsp70 amino acid phylogenetic analysis of B. tabaci and tomato genes are shown in Supplementary Figure S2.

To evaluate the ability of TRV-infected plants to express B. tabaci CypB and Hsp70 dsRNA and induce silencing in whiteflies, tomato plants were agroinfiltrated with the TRV::Bt CypB and TRV::Bt Hsp70 constructs separately. Agroinfiltration with TRV::Bt CypB (Figure 2B) and TRV::Bt Hsp70 (Figure 2C) did not affect tomato plant development, and no major visible morphological differences were observed between those plants and control plants (Figure 2A). Next, the effect of the expressed dsRNA of B. tabaci CypB and Hsp70 genes in tomato using TRV was tested on the whitefly development, gene expression and TYLCV transmission by feeding assays. Following feeding on pTV::Bt CypB, pTV::Bt Hsp70, TRV alone and mock inoculated plant leaves at three time intervals (24 h, 72 h, and 1 week), total RNA was extracted from both viruliferous and non-viruliferous whiteflies feeding on those plants and used in qRT-PCR. For Bt CypB, a gradual and significant down-regulation was observed from 24 h up to 1 week in non-viruliferous whiteflies (Figure 2D), while in viruliferous whiteflies a significant decrease in the expression was observed only at 72 h and 1 week compared to the levels at 24 h (Figure 2D). After Hsp70 silencing, significant decrease in the expression was observed after 1 week in non-viruliferous whiteflies, while a gradual significant decrease was observed at 72 h and 1 week after the start of the feeding in viruliferous whiteflies (Figure 2E). The levels of the transcripts of both CypB and Hsp70 genes remained stable in viruliferous and non-viruliferous whiteflies fed on the TRV1+TRV2 control plants (Figure 2D,E). Next, the virus levels in these experiments were also measured at the same time points where gene expression was measured. The results showed that the most significant effect on virus levels was observed in viruliferous whiteflies feeding on TRV::Bt Hsp70 dsRNA expressing plants after 72 h post whitefly release, while in the rest of the treatments the virus levels did not significantly decrease at the same magnitude, possibly due to virus degradation over time (Figure 3).

In the next experiments, we monitored whether the effects of gene silencing induced in the adults that fed on the dsRNA-expressing TRV plants, can be passed to the next generation and induce silencing and possibly phenotypic changes. Tomato plants agroinfiltrated with the pTV::Bt CypB, pTV::Bt Hsp70, TRV alone and mock inoculated plants were used for feeding adult whiteflies for 48 h after which they were transferred to cotton plants for egg lay. This transfer to cotton was performed to avoid any dsRNA acquisition by the next generation individuals from plants, and any observed effects would be attributed to dsRNA acquired from the previous generation. After a week, microscopic observations on the cotton leaves surfaces showed no difference in hatching between treatments and control insects. In all the agroinfiltrated groups, crawlers were observed over the leaf surface in search for a suitable settling site. Progeny collected from the pTV::Bt CypB, pTV::Bt Hsp70 showed phenotypic changes in second and third instar stages compared to TRV alone and mock infiltrated (Figure 4). Over all, different phenotypes were observed between nymphs fed on Bt CypB and Bt Hsp70 dsRNA-expressing plants. In the control insects, all instars looked oval in shape and flattened dorso-ventrally (Figure 4A,B,D,E). In case of nymphs feeding on CypB dsRNA-expressing plants, they appeared irregular in shape and eventually dried and fall off the leaves (Figure 4G,H). Interestingly, great effect on development at red-eyed nymph stage was noted and the red eyes were not observed as in normal development. Nymphs fed on pTV::Bt Hsp70 plants showed different phenotypic changes and the nymphs became flat and thin in size, edges of the nymph became more transparent and gradually dissolved on the leaf surface and dried (Figure 4J,K). We further examined the morphology of adults in the first generation of insects that have fed on dsRNA-expressing plants. In both TRV CypB- and TRV Hsp70 dsRNA-expressing plants on which the first generation was feeding, the adults exhibited curled winged morphology and lighter color of the body (Figure 4I,L), compared to control whiteflies that fed on control plants (Figure 4C,F). These whiteflies could not fly because of the wing morphology, hardly moved on the leaf, and died within 2–3 days without laying any eggs.

In these experiments, we quantified mRNAs levels in B. tabaci nymphs of the first generation that developed on the TRV-CypB, TRV-Hsp70, TRV alone and mock inoculated tomato plants using qRT-PCR in pools of second and third nymphal stages. The results showed dramatic suppression of the endogenous CypB and Hsp70 mRNAs (Figure 5). Quantification of the amount of CypB and Hsp70 genes were normalized against constitutively expressed B. tabaci α-tubulin levels. We observed ∼3 times depletion of CypB in nymphs when fed on TRV-CypB plants. Simultaneously, the expression of Cyp D and G genes, which were previously studied by us, was tested and found to be reduced by ∼2.5 and 1.5 times, respectively (Figure 5). These results imply that knockdown of CypB could affect other Cyp genes in the whitefly. In the case of Hsp70, mRNA levels were reduced by ∼4 times when fed on TRV-Hsp70, compared to the controls.

The effect of silencing CypB and Hsp70 on adult whitefly mortality and fecundity were examined. Experiments were performed over a period of 2 weeks in which adults were given access to TRV-silencing plants for both genes and the cumulative mortality and fecundity (number of laid eggs) were recorded. Surprisingly, we obtained very high mortality levels that reached 81.8% after feeding on TRV-CypB plants, and 85.6% when the adults were fed on TRV-Hsp70 plants, compared to the TRV and mock control plants where the mortality reached 20% and 16%, respectively.

Females that did not die following feeding on the TRV-silencing plants were recovered from all treatments. Ten females for each replicate were used for egg lay over a course of 2 weeks. The results showed that females feeding on TRV-CypB or TRV-Hsp70 plants laid 38.6% and 19.4% less eggs, respectively, compared to the control TRV and mock plants (Figure 6).

Some of the recovered females after feeding on silencing plants for both genes were used for virus transmission experiments. Adult MEAM1 females fed on TRV-CypB and TRV-Hsp70 expressing plants for a week or on TRV alone and mock agroinfiltrated plants as control, were caged with TYLCV-infected plants for a 48 h acquisition access period. Subsequently, single whiteflies were transferred to tomato plants in their three-leaf stage for 7 days inoculation access period. In the TRV-CypB and TRV-Hsp70 infected plants, 40% and 55% transmission rates were obtained on average, compared to ∼67% transmission rate when the whiteflies were fed on control plants (Figure 6).

B. tabaci is associated with bacterial endosymbionts that contribute to its successful biology and interactions with the environment (Gottlieb et al., 2008; Gueguen et al., 2010; Skaljac et al., 2017). Portiera is the primary endosymbiont while there were seven additional secondary symbionts reported from B. tabaci cryptic species around the world. The amounts and density of these different symbionts can be influenced from internal genetic factors in the whiteflies, as well as external environmental factors, which in turn can influence the whitefly biology (Brumin et al., 2011). In the current study, we tested whether the location and densities of the three endosymbionts Portiera, Rickettsia, and Hamiltonella that infect the MEAM1 species that we used in this study are influenced following the silencing of CypB and Hsp70. As seen in Figure 7, the location of all tested symbionts did not change between the control (Figure 7A,B,E,F), and insects that where fed on silencing plants (Figure 7C,D,G,H). Portiera and Hamiltonella colocalized inside the bacteriome cells, while Rickettsia localized outside the those cells, and occupied outside organs in the body cavity as previously reported (Gottlieb et al., 2008; Brumin et al., 2012). However, the most notable difference between the control and silenced insects was the shape and structure of the bacteriome cells. In the control insects, the cells looked intact and appeared as one structure, while in the insects in which CypB was silenced, the bacteriome looked elongated and the cells partially disintegrated (Figure 7C,G). In the insects in which Hsp70 was silenced, the phenotypes were even more severe where the bacteriome cells looked completely disintegrated, and the cells appeared all over the body when Portiera and Hamiltonella were localized (Figure 7H). When the densities of the three symbionts were measured after silencing using qPCR, the amounts of Portiera and Hamiltonella did not significantly differ compared with the controls, however, the amounts of Rickettsia were significantly reduced when CypB was silenced, a result that could be observed in the FISH results (Figure 7C).