Message t7 in vitro transcription kit

Three specific cDNA fragments of TaCIPK10 or TaNH2 with NotI and PacI restriction sites were obtained by reverse transcription PCR for VIGS analysis as previously described (Holzberg et al., 2002). In addition, we confirmed that the fragments showed no similarity with any other genes by BLAST analysis of the wheat genome databases (https://urgi.versailles.inra.fr/blast/). The capped in vitro transcripts of BSMV RNA were prepared from linearized plasmids containing the tripartite BSMV genome using the Message T7 in vitro transcription kit (Ambion, Austin, TX; Promega, Shenzhen, China) following the manufacturer's instructions. The more details of VIGS assay, host response detection and the methods of histological observation for fungal growth were performed according to a previous study (Liu et al., 2018).

To further determine the role of PsCPK1 during Pst infection, the PsCPK1 gene was silenced with the BSMV‐HIGS system. To make sure the specificity for PsCPK1 silencing, a 224‐bp fragment of PsCPK1 in the 5′UTR named PsCPK1‐1as and a 296‐bp fragment of PsCPK1 in the 3′UTR named PsCPK1‐2as were cloned with primers Higs‐CPK1‐1as‐F, Higs‐CPK1‐1as‐R, Higs‐CPK1‐2as‐F and Higs‐CPK1‐2as‐R (Table S3) and inserted into the virus plasmid. The BSMV RNAs were prepared in vitro from linearized plasmid γ‐TaPDSas, γ‐PsCPK1‐1as, γ‐PsCPK1‐2as, γ, α, β using the Message T7 in vitro transcription kit (Ambion, Austin, TX). The wheat leaves were used for inoculation with BSMV according to the procedures as previously described (Guo et al., 2013; Scofield et al., 2005). After inoculation with BSMV at the second leaf stage, wheat seedlings were maintained in a growth chamber at 23 ± 2 °C and examined for symptoms. In all experiments, the recombinant virus BSMV:TaPDSas was applied as a positive control. When the photobleaching phenotype was observed, the fourth leaves of PsCPK1 silencing group were inoculated with urediospores of Pst isolate CYR32. The resistant or susceptible phenotypes were visible at 15 dpi.