3.4. Transient Silencing of TaERFL1a

The functional complementation among homologs in the allohexaploid bread wheat genome hampers generation of a complete null cross [18]. To prevent functional complementation and allow complete silencing, we selected a conserved cDNA fragment (239 bp, +305 to +543 bp, translation start ATG is +1) of TaERFL1a, the three copies of which shared 98.4% similarities (Figure S2), possibly enabling simultaneous silencing by BSMV-VIGS. In addition, the selected fragment was outside the AP2/ERF domain to avoid silencing of other members of the ERF family (Figure S2). The primers and a schematic of the BSMV-VIGS-TaERFL1a vector are shown in Table S1 and Figure S7, respectively. Viral vector construction, viral RNA transcription, and viral inoculation for BSMV-VIGS were performed as described previously [41,42]. BSMV-VIGS-GFP-inoculated wheat seedlings were used as the negative control, and the appearance of chlorosis on the inoculated tissues confirmed successful BSMV inoculation and target gene silencing in the host plant [43].

The RNA-α, RNA-β, and RNA-γ transcripts of the BSMV-VIGS-TaERFL1a or BSMV-VIGS-GFP vectors were mixed in a 1:1:1 ratio and diluted with nine volumes of diethyl pyrocarbonate (DEPC)-treated water. Next, 12 volumes of 2× GKP buffer (50 mM glycine, 30 mM, pH 9.2 dipotassium hydrogen phosphate, 1% bentonite, 1% Celite) were added. Virus inoculation was performed on the second leaves of wheat seedlings (Yumai 34 cultivar), and was accomplished by gently rubbing the leaf surface five times from the central section of wheat leaves to the tip and then from base to tip [42]. At 8 days after inoculation, the phenotypes of inoculated wheat seedlings were assessed and the TaERFL1a transcript levels were measured by qPCR. Subsequently, wheat seedlings inoculated with BSMV-VIGS-TaERFL1a and BSMV-VIGS-GFP transcript virus were separately transferred to full-strength Hoagland solution with 20% PEG6000 for 3 days. The fresh weights, dry weights, absolute water contents, and MDA concentrations of the wheat seedlings were determined as described previously [44].