Attempt at silencing HvPht1;1 expression in barley roots. A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of "USER cloning" BSMV vectors are longer by 22 bp compared with restriction enzymes cloning. Not drawn to scale. C Stability of the BSMV-Pht1;1 and BSMV-GFP375 constructs in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert as shown in B. Lanes 1, 2, 3: BSMV-Pht1;1, 0 mM Pi (610 bp); 4, 5, 6: BSMV-Pht1;1, 1 mM Pi (610 bp); 7, 8, 9: BSMV-GFP375, 0 mM Pi (617 bp); 10, 11, 12: BSMV-GFP375,1 mM Pi (617 bp); 13: plasmid containing BSMVγ-PDS cassette (643 bp); 14: water control; M2: DNA marker; black arrow represents DNA fragment of 564 bp. The expected lengths for PCR products are given in brackets.