VIGS Cloning and Propagation

All VIGS procedures were adapted from Lee et al. (2015) with minor changes. In short, a 250–400 bp segment was designed for each gene using the si-Fi (siRNA Finder)¹ software, based on the “Zavitan” WEW transcriptome. Anti-sense sequences were amplified from Zavitan genome using specific primers (Supplementary Table 1) and cloned into the BSMV RNAγ vector pCa-γbLIC (Yuan et al., 2011) via ligation-independent cloning (LIC) and transformed into Agrobacterium tumefaciens strain GV3103 as described (Lee et al., 2015). Four weeks old Nicotiana benthamiana leaves were co-infiltrated with a mix of Agrobacterium tumefaciens strains carrying BSMV RNAα, RNAβ, and RNAγ together in 1:1:1 ratio. Infected leaves were collected 5 days post infection and either stored at −80°C for later use or were used immediately for wheat infection. Non-infiltrated leaves were collected 8 days post infection to verify systemic infection ability of the virus. To that end, total RNA was purified using Nucleospin RNA Plant kit (MACHEREY-NAGEL) followed by cDNA synthesis with Verso cDNA synthesis kit (Thermo Fisher Scientific). Viral presence was verified using primers from the virus genome and the specific insert (Supplementary Table 1).