Construction of BMV VIGS Vectors

PCR primers for cloning of TaPHO2 and TaPDS fragments from wheat (cv. Overley) were designed at the conserved regions of the three homoeologs for each gene. The 52-, 100-, 150-, 200-, and 250-bp fragments of TaPDS (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"FJ517553.1","term_id":"219814634","term_text":"FJ517553.1"}}FJ517553.1) were amplified by RT-PCR using primer pairs of P1-F4 /P1-R2, P1-F4/P1-R1, P1-F3/P1-R1, P1-F2/P1-R1, and P1-F1/P1-R1, respectively. The 114-, 150-, 204-, and 252-bp fragments of TaPHO2 (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"AK331438.1","term_id":"241983498","term_text":"AK331438.1"}}AK331438.1) were amplified using primer pairs of P2-F1/P2-R4, P2-F1/P2-R3, P2-F1/P2-R2, and P2-F1/P2-R1, respectively. Similarly, The 107-, 180-, and 220-bp eGFP (GenBank accession: {"type":"entrez-nucleotide","attrs":{"text":"U55761.1","term_id":"1377908","term_text":"U55761.1"}}U55761.1) fragments were amplified from the T3 transgenic OsRCg2GFP wheat seedlings using primer pairs P3-F3/P3-R1, P3-F2/P3-R1, and P3-F1/P3-R1, respectively. All forward primers and reverse primers contained, respectively, an AvrII (CCTAGG) or NcoI (CCATGG) restriction enzyme site. The resulting PCR fragments were cloned into the AvrII and NcoI sites of the pC13/F3CP5, encoding RNA3 of the BMVCP5 vector. The pC13/F3CP5 plasmid with pC13/F1+2, encoding RNAs 1 and 2 of BMV, composed the BMVCP5 vector and were used in the VIGS experiments. BMV:GFPuv, a BMVCP5 vector with a 250-bp fragment from a variant of the GFP gene (Ding et al., 2018), was used to screen susceptibility of wheat cultivars for BMV infection. Primers used for construction of BMVCP5 VIGS vectors in this study are listed in Supplementary Table 2.