Figure 2.

Stability of insert fragments and gene silencing in wheat leaves with BMVCP5. RT-PCR products from wheat leaves of plants inoculated with BMVCP5 harboring gene fragments in length from 100 to 252 nt from eGFP (A), TaPDS (B), and TaPHO2 (C). Subscript numbers after target gene designation indicate the insert size in the BMV vector. The 1st or 2nd inoculated leaf or 3rd systemic leaf were harvested, respectively, at 4, 7, and 20 dpi, and total RNA extracted for RT-PCR amplification using primers flanking the cloning site (P4-F/P4-R). Each lane represents an RT-PCR product from an individual plant. Similar results were obtained in three independent experiments. A 1 kb plus DNA ladder (M, Invitrogen) and a 427-bp PCR product amplified from plasmid pC13/F3CP5 with no insert (EV) serve as size markers. (D–F) Relative expression levels of target gene mRNA in the 3rd systemic leaf at 20 dpi were determined by RT-qPCR using the same cDNA analyzed for insert stability in Panels (A–C) and primers specific for the host target mRNA (but not the relevant insert fragment). PCR product quantities for target genes were normalized against the levels of wheat translation elongation factor subunit EF1α (TaEF1α) mRNA. (D) Relative expression levels of TaPDS mRNA in plants infected with BMV:eGFP₁₈₀, BMV:TaPDS₁₀₀ or BMV:TaPDS₂₅₀. (E) Relative expression levels of TaPHO2 mRNA in plants infected with BMV:eGFP₁₈₀, BMV:TaPHO2₁₁₄ or BMV:TaPHO2₂₅₂. (F) Relative expression levels of TaPHO2-A1, TaPHO2-B1, TaPHO2-D1 mRNA in plants infected with BMV:eGFP₁₈₀ or BMV:TaPHO2₁₁₄. Values in panels (D–F) represent means + SE of four or five biological replicates. Significant differences between treatment mean values in panels (D,E) are indicated by different letters above bars for each treatment (P < 0.05, significant ANOVA followed by LSD analysis); treatment values in panel (F) were compared by t-test (**P < 0.01). Percentage values are relative to the BMV:eGFP₁₈₀ control.