In vitro synthesis of dsRNA
Total RNA was extracted from neonate EAB larvae using TRIzol reagent (ThermoFisher, USA). The quality and quantity of the RNA were checked by electrophoresis and spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). cDNA was synthesized from 1 µg of total RNA using a M-MLV reverse transcriptase kit (ThermoFisher, USA).
The PCR templates for in vitro synthesis of dsRNA were generated using gene-specific primers containing T7 polymerase promoter sequences. PCR conditions were 4 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 45 s at 72 °C, finishing with an extension step at 72 °C for 10 min. The PCR template was purified using a PCR purification kit (Qiagen Inc., Valencia, CA, USA). After PCR purification, dsRNA synthesis was performed using the MEGAscript RNAi Kit (Ambion Inc., Foster City, CA, USA) following the manufacturer’s instructions. The reaction was incubated for 14 h at 37 °C, followed by 15 min of DNase treatment. The dsRNA was precipitated by adding 0.1 × volume of sodium acetate (3 M, pH 5.2) and 2.5 × the volume of 100% ethanol and kept at − 20 °C for at least 2 h followed by centrifugation at 4 °C and 14,000g for 30 min. The dsRNA pellet was then rinsed with 750 μl of 75% ethanol and centrifuged again at 4 °C for 15 min. The ethanol was removed and the dsRNA was diluted in ultrapure distilled water. The quality of the dsRNA was checked by electrophoresis and quantified using a spectrophotometer (NanoDrop Technologies, Wilmington, DE USA). To attain the desired concentration for each assay, dsRNA samples were vacuum concentrated using Concentrator plus (Eppendorf, Hauppauge, NY, USA).
To evaluate dsRNA uptake through plant and insect tissue, dsRNAs were fluorescently labeled using the Silencer siRNA Cy™3 labeling kit (ThermoFisher, USA) with minor modifications to the manufacturer’s protocol. dsRNAs were labeled by adding Cy3 labeling reagent and incubating for 3 h at 37 °C. Labeled dsRNA was precipitated with 0.1 volume of NaCl and 2.5 volumes of 100% ethanol followed by incubation at − 20 °C for 30 min. Precipitated labeled dsRNA was recovered by centrifugation at 4 °C and 14,000g for 20 min, and the pellet was further washed with 70% ethanol. The recovered pellet was air-dried and re-suspended in nuclease-free saline. The concentration of labeled dsRNA was determined using a spectrophotometer (NanoDrop Technologies, Wilmington, DE USA).
dsRNA absorption by plant tissue
Green ash, F. pennsylvanica, seedlings ~ 5 cm in length (N = 2) were immersed in a 5-ml beaker containing labeled dsSHI (Rodrigues et al. 2017b) (70 µg/ml) in 2 ml RNase and DNase-free water (Table 1a), covered with parafilm, and incubated at room temperature (~ 21 °C) for 48 h to allow capillary action to draw the solution to the top of the plants. Samples were washed with deionized water, and total RNA was extracted from root, stem, and leaf tissue, and cDNA was synthesized from 1 µg of total RNA using a M-MLV reverse transcriptase kit (ThermoFisher, USA). The cDNA was subjected to PCR validation using primers specific to dsRNA (Rodrigues et al. 2017b). PCR conditions were 4 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, and 45 s at 72 °C, finishing with an extension step at 72 °C for 10 min.
Greenhouse-grown tropical ash, F. uhdei, were used to evaluate dsRNA uptake (Table 1b). Branches (~ 1 cm diameter, N = 5) were excised and immediately placed into 5-ml plastic test tubes containing 1 ml of a labeled dsGFP solution, covered with parafilm, and maintained at room temperature (~ 21 °C). The dsRNA concentration was 20 μg/ml resulting in a final exposure of 20 μg of dsRNA per branch. After the initial 1 ml of labeled dsGFP was taken up by the plant tissue, 500 µL of water was added to the tubes every other day.
dsRNA uptake by insect tissue
Recovered EAB neonate larvae
Ingestion of dsRNA-treated ash tissue by developing neonate larvae (Table 1c) was evaluated using the same greenhouse-grown tropical ash described above. Branches were artificially infested with laboratory-reared EAB eggs (Olson and Rieske 2019) prior to branch excision. Briefly, pieces of coffee filter (Kroger, Cincinnati OH) with laboratory-reared EAB eggs laid on them were attached to branches and secured using 1.5-cm-wide strips of parafilm (Bernis NA, Neenah, WI). Additional eggs (N = 5–8) were kept under the same conditions, but in Petri dishes with moistened filter paper, to help identify the hatching time. When the eggs in the Petri dish started hatching, the branches were excised and treated with dsRNA as described above. There were five branches per treatment (labeled dsGFP and water), each one artificially infested with six EAB eggs. After 8 days the treated branches were carefully debarked using a sharp knife, and EAB egg hatch, larval development, and phloem consumption were evaluated (Olson and Rieske 2019); confocal microscopy was used to evaluate labeled dsRNA uptake.
EAB egg chorion
To evaluate the uptake of dsRNA through the egg chorion (Table 1d), ~ 15 laboratory-reared EAB eggs were placed in Petri dishes (60 × 9 mm) with moistened filter paper and soaked with 1 μl of labeled dsGFP at a concentration of 1 μg/μl. Water was used as the negative control. The dsRNA treatment was repeated 3 times over 48 h, during which the eggs were kept at 23 °C and 75% relative humidity in a growth chamber. After 3 days the eggs were evaluated under confocal microscopy to assess labeled dsRNA uptake; confocal microscopy was used to evaluate labeled dsRNA uptake.
Confocal laser scanning microscopy
All tissues were imaged using an Olympus FV1200 confocal laser scanning microscopy (Center Valley, PA, USA). The fluorescence of Cy3 in the tissues was detected using a 559-nm laser with a bandpass filter of 570/100 nm. ImageJ software was used for image analysis. Fluorescently labeled samples and control samples, treated either with unlabeled dsRNA or water, were imaged simultaneously using the same parameters to ensure minimal background fluorescence in the negative controls and the positive signals for direct comparison.
After 48-h exposure to labeled dsRNA, plants were sectioned into stem (1 mm cross section), root (simply separated from the aerial part), and leaf (1 × 1 cm square) tissues using a razor blade and imaged using confocal microscopy under the conditions described above.
Ash branches and recovered EAB neonate larvae
After 8 days of exposure to labeled dsRNA, branches were carefully debarked, EAB neonate larvae were recovered, and the branches were sectioned. Bark, ash stems, and EAB larvae recovered from treated twigs were imaged using the instrumentation and methods described above.
EAB egg chorion and neonate larvae
After 48-h exposure to labeled dsRNA, EAB eggs were imaged using the same method described above. Similarly, eggs that had been treated with the labeled dsRNA were allowed to hatch and the neonate larvae were imaged after 48 h.
Adult insect assays
Leaves of greenhouse-grown tropical ash containing 3–5 leaflets were excised with petioles intact and immersed in 2 ml of water containing 70 µg/ml of EAB-specific dsSHI for 2 h to allow uptake; dsGFP was used as the control (Table 1e). Laboratory-reared adult EAB (N = 5–10) was then placed on dsRNA-treated leaves and allowed to feed for 10 days (Fig. 1). Assays were held in ventilated plastic cups (500 ml) and maintained in a growth chamber at 25 °C and 16:8 L:D. Each treatment was replicated three times, and beetle survival was recorded daily. Mortality (%) was calculated, and the mean value of the experimental replicates was analyzed using a one-way ANOVA, followed by Tukey’s post hoc t test. In addition, in a fourth replicate, adult EABs (N = 5–10) were collected at 24 h, 72 h, and 120 h following exposure to dsRNA-treated leaves, and total RNA was isolated for gene expression analysis.
Quantitative real-time PCR analysis
The levels of transcript expressed by eggs, neonate larvae, and adults after exposure to dsRNA were measured by quantitative real-time PCR (RT-qPCR). Following total RNA extraction, cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies, Carlsbad, CA, USA) and used as a template for gene expression studies. The expression analyses were conducted using SYBR Green PCR Master Mix. The PCR mixture contained 1 μl of cDNA, 0.2 μl of each primer (10 mM), 5 μl of the SYBR green PCR master mix, and 3.6 μl of ddH2O, totaling 10 μl. Real-time quantitative RT-PCR (RT-qPCR) was performed using the StepOnePlus Real-Time PCR system (Life Technologies, Carlsbad, CA, USA) under the following conditions: one cycle of 20 s at 95 °C, followed by 40 cycles of denaturation at 95 °C (3 s), annealing, and extension for 30 s at 60 °C, ending with generation of a melting curve to confirm a single peak and rule out nonspecific product and primer dimer formations. The reference gene used was TEF1α (Rajarapu et al. 2012; see Supplemental Materials), and the 2-ΔΔCt method was used to calculate the relative expression of the target gene compared to the control (Livak and Schmittgen 2001). A two-tailed t test was used for statistical analysis to compare the means of a single variable.