Figure 7

Analysis of the transgene re-arrangments in Fusarium oxysporum lines S34 and S5. (A) Schematic diagrams (not to scale) of the transgenes in F oxysporum lines S34 and S5. The structure of the S34 locus was determined by sequencing of a lambda phage clone containing this entire region and was derived likely by recombination between the pre-existing Gus transgene and an incoming hpGus transgene, such that the full length Gus ORF is followed by the 550 nt antisense Gus arm and the gpdA promoter, both derived from the hpGus transgene. The resulting hairpin-like Gus sequence is flanked by convergent gpdA promoters. Details of both transgenes prior to the recombination event are shown in Figure 1. Fox, F. oxysporum genomic sequences; λT3 and λT7, lambda phage T3 and T7 RNA polymerase binding sites. (B) The hpGus transcripts in strain S5 were likely derived from an endogenous promoter 3′ of the T-DNA insertion site. Total RNA (10 μg) from the Gus 0–1.6 parent (left lane), 0–1.6 hpGus lines (middle two lanes) and WT hpGus lines (right two lanes) was hybridized with a probe detecting antisense trpC terminator sequences. TrpC antisense sequences were only present in line S5, suggesting that these transcripts are produced by an endogenous promoter located downstream of the hpGus integration site. (C) Hybridization of total RNA (10 μg) with an antisense Gus probe specific for the loop region of the hpGus transgene, detecting transcripts that contain sense Gus sequences. Transcripts derived from the resident Gus transgene (1.8 kb) were detected in all samples except S5, which does not carry the Gus transgene. The 1.1 kb Gus sequence detected only in RNA of S5, corresponds to the hairpin loop region, likely produced by dicer processing of a correctly folded hairpin transcript.