BSMV-VIGS for Silencing Wheat Genes

Target fragments for silencing (V1 and V2) that had no or low similarity with other genes and thus unlikely to cause off-target effects were selected based on predictions made using si-Fi software (http://www.snowformatics.com/si-fi.html). The plasmids (γ-V1 and γ-V2) utilized for gene silencing were constructed according to the methods described by Holzberg et al. (2002) (link). Plasmids α, β, γ, γ-PDS, γ-V1, and γ-V2 were each linearized. The linearized plasmids were used as templates for in vitro transcription using the mMESSAGE mMACHINE^® Kit High Yield Capped RNA Transcription Kit (Ambion) according to the manufacturer’s protocol. BSMV VIGS vectors harboring target gene sequences and Pt urediospores were co-inoculated into wheat cultivars TcLr19 and Thatcher according to previously described methods (Wang et al., 2020 (link)). Plants mock inoculated with sterile water and plants inoculated with a VIGS vector targeting the wheat phytoene desaturase (TaPDS) gene were used as negative and positive controls, respectively.