Quantifying Aphid Gene Expression

Bacteria were delivered to A. pisum LSR1 in artificial diet as described in the previous methods sections. For these experiments, five conditions were prepared: CWBI-2.3^T-GFP + BTK-NR, CWBI-2.3^T-GFP + dsE2C-800, CWBI-2.3^T-GFP + dsC002-800, CWBI-2.3^T-GFP + dsE2C-800 and CWBI-2.3^T + dsNuc1-800, and CWBI-2.3^T-GFP + dsC002-800 and CWBI-2.3^T + dsNuc1-800. Two sets of 20 aphids were treated to each condition; the aphids were then transferred to plants after 24 h of feeding. Five days after treatment, 6–10 aphids colonized with fluorescent bacteria from each condition were collected and crushed in DNA/RNA shield (Zymo Research, Irvine, CA, USA). Whole aphid RNA was purified using an RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) which includes an on-column DNA digestion step. Five hundred nanograms of each RNA sample were then reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Waltham, MA, USA). Primers for qPCR were designed using IDT’s Primerquest tool and are listed in Table S4. All qPCR reactions were prepared in triplicate in 384-well plates with SYBR Green reagents (Thermo Fisher, Waltham, MA, USA), and reactions were carried out using a ViiA-7 Real-Time PCR System (Thermo Fisher, Waltham, MA, USA).
Control qPCRs were run to determine the efficiency of each primer set and establish the most reliable reference genes for analysis. To determine primer efficiency, PCR products for each qPCR primer pair were synthesized, serially diluted, and run as the template to generate a standard curve. To choose the most reliable reference genes, we tested Beta-tubulin, GAPDH, NADH, Rpl32, and SDBH. We selected SDBH and Rpl32 to serve as references because they had the lowest variance between conditions. We calculated the gene expression ratio for each aphid target relative to the geometric mean of both reference genes by the Pfaffl method (Pfaffl, 2001 (link)). The amount of dsRNA production was determined by running the cDNA samples along with a standard curve for the gene of interest. The Cq values returned for the standard curve were then used to calculate the absolute copy number for each condition (Bustin, 2000 (link)).