Figure 5

Endogenous genes can be targeted by hpRNA-derived small interfering RNAs (siRNAs). Wild type (WT) Fusarium oxysporum was transformed with a hpRNA construct directed against the endogenous Frp1 gene. Total RNA (15 μg) from nine independent transgenic lines was separated on a 17% polyacrylamide gel and hybridized with a probe specific for Frp1. The hpFrp-derived siRNAs were detected in most lines although the levels are very low (upper panel). The U6 transcripts are shown as loading control. Total RNA (10 μg) was separated on an agarose gel and hybridized with an Frp1 sense probe to detect antisense sequences of the hpFrp transgene (middle panel). To detect Frp1 mRNA levels, total RNA (10 μg) was hybridized with a probe specific for the 3′ region of the endogenous Frp1 gene, which is not present in the hpFrp gene, detecting 2.3 kb Frp1 mRNA, but not hpFrp transcripts (lower panel). Ethidium bromide-stained ribosomal RNA is shown as loading control. The additional transcripts detected are likely to be either Frp1 mRNA cleavage products (below the endogenous transcript band) or size mobility shifted endogenous Frp1 likely due to binding of small RNAs (above the endogenous transcript band), as both are not present in the WT sample.