Mmessagemmachine t7

Plasmids for DENV or ZIKV vectors which encode GFP (pFK-DVs-G2A for DENV_GFP), Renilla luciferase (pFK-DVs-R2A, serotype 2, strain 16681 for DENV_R2A; pFL-ZIKV-R2A strain FSS13025 for ZIKV_R2A) reporter genomes, wild-type (WT) DENV (pFK-DVs; 16681), subgenomic (sg) replication-deficient NS5 mutant (sg-DVs-R2A-GND), and WT (sg-DVs-R2A-WT) subgenomic replicon systems were all obtained from Ralf Bartenschlager and Pei-Yong Shi. Plasmids were linearized with XbaI (DENV) or ClaI (ZIKV) and purified, and 1 μg was in vitro transcribed using the mMESSAGE mMACHINE T7 or SP6 transcription kit (Invitrogen). The resulting RNA quality and concentration were assessed by migration on a 0.8% agarose gel and Nanodrop spectrometry (Thermo Scientific), respectively.

To silence the corresponding TaANKTM genes, fragments of the three selected TaANKTM2A-5/TaANKTM3A-2/ TaANKTM6A-1 genes with the length of 234 bp, 242 bp, and 276 bp were amplified with corresponding primer pairs (Supplementary Table 1). Each of the target fragment was inserted into the γ-strain of BSMV by the homogenous recombination method to produce BSMV:TaANKTM vectors. The second fully expanded leaves of AK58 were infected with the in vitro transcribed (mMESSAGEmMACHINE T7, Invitrogen, Waltham, MA, USA) viruses BSMV:TaANKTM2A-5, BSMV:TaANKTM3A-2 and BSMV:TaANKTM6A-1, while seedlings infected with BSMV:TaPDS and BSMV:γ served as controls. The infected plants were grown at 23°C, with a 14 h light/10 h dark cycle environment condition with 70% relative humidity. The fourth fully unfolded leaves with visible viral infection symptoms were detached and placed on 6BA-plate with mixed race of Bgt spores to evaluate disease resistance. The inoculated leaves were cultured in a light incubator with a cycle of 14 h light/22°C and 10 h dark at 18°C for 6 days. Target genes silencing efficiency were evaluated by qRT-PCR using the corresponding primer pair TaANKTM-Q (Supplementary Table 1).

The target gene of tae-miR397 was predicted to be TraseCS6A02G134500 using psRNATarget (http://plantgrn.noble.org/psRNATarget/, 18 March 2022) and named Tae-WIP. TaeWIP has three copies scattered in 6A, 6B and 6D genomes, including TraseCS6A02G134500, TraseCS6B02G162700 and TraseCS6D02G123700. Because of the high sequence similarity of the three copy genes in different genomes, we cloned the common sequence of the three genes for silencing. The BSMV-VIGS system was used to knock down the transcript level of the target gene. The BSMV-mediated gene silencing in wheat leaves was carried out as described by Yuan et al., 2011 [66 (link)]. The 277 bp fragment of the target gene (from +66 to +342) was amplified using primers and cloned into the BSMV:γ vector via homogenous recombination to generate the recombination vector BSMV:WIP. BSMV:TaPDS and BSMV:γ vector were employed as controls. The fully expanded second leaves of the wheat Bainong207 were infected with vitro-transcribed (mMESSAGEmMACHINE T7, Invitrogen, Waltham, MA, USA) viruses BSMV:WIP, BSMV:TaPDS and BSMV:γ. The completely expanded fourth leaves with apparent virus feature after 15 days of infection were collected for inoculation with Bgt, and the measurement of silencing efficiency was performed using qRT-PCR. The phenotype of disease symptoms was recorded at 7 dpi. All primers are listed in Supplementary Table S1.