Bacterial Conjugation and RNAi Delivery

Construction of CWBI-2.3^T-GFP was described previously (Elston et al., 2021 (link)). HT115-GFP was built using pGRG36::PA1-GFP, a plasmid derived from pGRG-36 (Phillips & Cooper, 2021 (link)). To apply this system to HT115, we adapted methods from McKenzie & Craig (2006) (link). The donor E. coli strain, MFDpir, and the recipient HT115 strain were first grown overnight, then washed twice with 145 mM NaCl saline solution, mixed to a 1:2 ratio of donor:recipient, and 50 µL of the mix was spot-plated on LB + DAP agar. After a day of growth, the spot was scraped up and washed with saline as before. Dilutions of these bacteria were plated on LB + Carb agar and grown at 30 °C to select for transconjugants that had acquired the plasmid. Then, colonies were picked and re-grown on LB + Cam agar at 37 °C to select for transposition of the GFP cassette into the chromosome and loss of the temperature-sensitive plasmid. The resulting colonies were picked and regrown at 37 °C in liquid LB with Cam or Carb to confirm loss of the plasmid and successful integration.
The design of our dsRNA expression vector is based on a system for smRNAi in honey bees (Leonard et al., 2020 (link); Lariviere et al., 2022 (link)). In brief, the plasmid has a low to medium copy RSF1010-based origin of replication, spectinomycin resistance, and a dsRNA target sequence flanked by two identical inverted CP25 promoters. Each target for the RNAi experiments was first amplified from A. pisum LSR1 cDNA with “GGA” primers specified in Table S2, then cloned into the dsRNA site on the pBTK800 expression vector (Lariviere et al., 2022 (link)) with BsaI Golden Gate Assembly. The choice of amplified gene fragment was selected to replicate previous publications (Mutti et al., 2006 (link); Vellichirammal et al., 2017 (link); Chung et al., 2018 (link)). Assembled plasmids were conjugated or electroporated into CWBI-2.3^T-GFP and HT115-GFP for aphid feeding as described previously (Elston et al., 2021 (link)), except the dsNuc1-800 plasmid, which was tested in wild-type CWBI-2.3^T and HT115 hosts. Plasmids used in this study are shown in Table S3.