Figure 4

Evaluation of HvCEBiP gene silencing. (A) The genomic organization of BSMV and corresponding silencing constructs. Genomic RNA of BSMV was transcribed in vitro from pα42, pβ42.sp1 and pSL038-1, carrying the α, β and γ genomes, respectively. Genomic RNA of BSMV:HvCEBiP was from pα42, pβ42.sp1 and pγ:HvCEBiPas, which harbours a partial fragment of HvCEBiP in the antisense orientation. (B) The third-developed leaves of barley plants at 10 days after inoculation with BSMV genomic RNA onto the first leaves. Stripe mosaic symptoms were observed in the third leaves of BSMV- or BSMV:HvCEBiP-treated plants but not in untreated plants (No BSMV). (C) Evaluation of the silencing effect by RT-PCR. Total RNAs were extracted from the leaves shown in B and used for RT-PCR. BSMVCP encoding viral coat protein was detectable in BSMV- or BSMV:HvCEBiP-treated plants but not in untreated plant (No BSMV). The expression level of HvCEBiP was down-regulated in the third leaves of BSMV:HvCEBiP-treated plants compared to a BSMV-treated plant or untreated plant. For checking genomic contamination, PCR of HvEF1α was performed using total RNA as template (RT-). Ribosomal RNAs are presented as loading control.