RNA Interference to Investigate Insect Virus Transmission

RNA inference was performed to knock down the expression of related genes. The T7 promoter with the sequence 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGG-3′ was added to the forward and reverse primers at the 5′ terminal to amplify a region of ~500–800 bp of HongrES1, RdFV CP, RGDV P8, or GFP gene (Supplementary Table 1). The PCR products were used for the synthesis of dsRNAs targeting HongrES1 (dsHongrES1), RdFV CP (dsCP), RGDV P8 (dsP8), or GFP (dsGFP) according to the protocol for the T7 RiboMAX Express RNAi System kit (Promega, P1700).
To test the knockdown of HongrES1 expression on RdFV or RGDV infection in male reproductive systems, newly emerged male adults of RdFV-positive or RGDV-positive R. dorsalis population were microinjected with dsHongrES1 or dsGFP (approximately 200 ng/leafhopper) using a Nanoject II Auto-Nanoliter Injector (Spring), and then transferred to healthy rice seedlings. To test the knockdown of RdFV CP or RGDV P8 expression on viral infection and HongrES1 accumulation in male reproductive system, newly emerged male adults of RdFV and RGDV co-positive R. dorsalis population were microinjected with dsCP, dsP8 or dsGFP (~200 ng/leafhopper), and then transferred to rice seedlings. For each treatment, approximate 100 insects were microinjected, and three replicates were performed.
The male reproductive organs were dissected to test the expression levels of RdFV CP, RGDV P8, or HongrES1 using RT-qPCR and western blot assays. A pool of 30 dsRNA-treated males was used for each replicate, and the experiment was conducted in three replicates for RT-qPCR assays. The total proteins from reproductive organs of 30 dsRNA-treated males were analyzed for the protein levels in western blot assays by using HongrES1-, CP- or P8-specific IgG (0.5 μg/μl). Experiment was conducted in three replicates in western blot assays. To determine the effect of dsHongrES1, dsCP or dsP8 treatment on paternal transmission of RdFV or RGDV, one dsHongrES1-, dsCP-, dsP8- or dsGFP-treated RdFV- or RGDV-positive male mated with one virus-free virgin female in a glass tube containing a rice seedling for 3 days (Supplementary Table 3). Ten pairs were performed for each treatment. The males were then tested for presence of RdFV or RGDV, and females were left in the tubes for oviposition. The offspring of each mating combination were tested for presence of RdFV or RGDV by RT-PCR assays. The primers used in RT-PCR assays were shown in Supplementary Table 1. The experiment was conducted in three replicates.