Detection of CesA antisense siRNAs by ribonuclease protection assay. (A) Ribonuclease protection assays performed with the short HvCesA6 sense probe were hybridized with 10 μg yeast RNA (negative control), A6-sense (probe-only, no RNase control), coleoptile RNA (10 μg), PDS-only (10 μg Vector-PDS VIGS RNA), PDS-CesA-CR1 (10 μg CesA-CR1 VIGS RNA), and PDS-CesA-CR2 (10 μg CesA-CR2 VIGS RNA). The vector-only probe [vector; probe-only controls without RNase were hybridized with 10-μg yeast RNA (negative control) and barley leaf (10-μg barley third leaf RNA). (B) Protection assays were also performed with the short HvCesA6 sense probe hybridized with barley third leaf RNA (10 μg each) treated with Mock buffer (C), 100-μM DCB (+DCB), or 1-μM isoxaben (+ISX). Decade markers (Ambion) were electrophoresed as size standards and are to scale for both (A) and (B). Large arrowheads denote 21-nt RPA fragments, and the small arrowheads denote the 24-nt RPA fragments. Marker lanes are in 10-nt increments beginning with 20 nt.