Mmessage mmachine kit high yield capped rna transcription kit

In order to confirm the specificity of the silenced fragments, fragments were selected that had no or lower similarity with other genes to prevent off-target, then one cDNA fragments of TaTLP1 and two cDNA fragment of TaPR1 for gene silencing with Not I and Pac I restriction sites (S3 Table) were obtained by reverse transcription PCR (RT-PCR) and inserted into the original barley stripe mosaic virus (BSMV) vector [49 (link)]. In vitro transcription was performed using the mMESSAGE mMACHINE Kit High Yield Capped RNA Transcription Kit (Ambion) with linearized plasmid as template according to the manufacturers protocol. Then 240 μl of the BSMV mixture was applied to fully expanded leaves of 2-leave stage TcLr19 wheat plants by rubbing according to the method described previously [50 (link)]. Leaf rust urediniospores were inoculated on the fourth and fifth leaves of TcLr19 and the susceptible wheat cultivar Thatcher. Sterile water was inoculated to wheat leaves as control. The more details of VIGS assay were performed according to a previous study [24 (link)]. The feasibility and silencing efficiency were tested using the wheat phytoene desaturase (TaPDS) as a positive control.

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.