Wheat TaCDPK27 Silencing via BSMV-VIGS

The BSMV-VIGS method was performed to acquire TaCDPK27-silenced wheat seedlings as previously described [48 (link)]. A cDNA fragment of 131 bp (+1612 bp to +1742 bp) was employed to acquire the TaCDPK27-silenced vector. After this fragment was inserted into the BSMVγ plasmid (BSMVγ:TaCDPK27), the plasmids BSMVα, BSMVγ:GFP and BSMVγ:TaCDPK27 were linearized with Mlu I (Takara, Dalian, China). BSMVβ was linearized with Spe I (Takara, Dalian, China). Then, a RiboMAX large-scale RNA Production-T7 kit (Promega, Madison, WI, USA) was used to transcribe these linearized plasmids in vitro. Ribom 7G Cap Analog (Promega, Madison, WI, USA) was employed to produce the 5′-capped BSMV RNA molecules for subsequent BSMV-VIGS inoculation experiments. These experiments (including vector construction, in vitro transcription, BSMV-VIGS inoculation, TaCDPK27-silenced seedling identification, and silencing efficiency assessment of TaCDPK27) were conducted as previously described [45 (link)]. Then, the BSMV-VIGS-inoculated seedlings with three fully expanded leaves were treated with 150 mM NaCl for 6 days. All experiments had at least three separate replications. The primes used are listed in Table 3.