Feeding assays and RNA isolation

The young first instar nymphs of S. graminum and R. padi were fed on BYDV-infected, WDV-infected, and healthy wheat plants to achieve six experimental treatments: the vector treatment was defined as S. graminum fed on BYDV-infected wheat (Sg-BYDV); the non-vector treatment was defined as S. graminum fed on WDV-infected wheat and R. padi fed on BYDV-GAV-infected, WDV-infected wheat (Sg-WDV, Rp-BYDV, Rp-WDV); and the control treatments were S. graminum and R. padi fed on healthy wheat seedlings (Sg-ck, Rp-ck).

Three biological replications of each treatment were conducted and each replicate contained 20 individual aphids. For each treatment-replicate, they were maintained in a growth chamber under the same environmental conditions as described above. We collected 20 adult aphids into a 1.5 ml tube, and they were immediately flash frozen in liquid nitrogen and stored at − 80 °C. Total RNA was extracted from each sample using TRIzol reagent (TaKaRa, Japan) following the manufacturers’ instructions. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using a Qubit® RNA Assay Kit in a Qubit®2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).