Expression analysis of the hydrophobin genes bhp1 , bhp2 and bhp3 , and six hydrophobin-like genes. A: Results of semi-quantitative RT-PCR, showing gene expression in different developmental stages of wild type B05.10, the hydrophobin triple mutant Δbhp3/bhp1/bhp2, and the Δbhl1 mutant (lanes with cDNA from Δbhl1 labelled with stars). M: Size markers, with relevant sizes indicated [bp]; W: Water control; G: Genomic DNA; Co: Resting conidia; My: mycelium (15 h.p.i.); To: Infected tomato leaves (48 h.p.i.); Sc: Sclerotia; Fr: Fruiting bodies. An EF1α encoding fragment was amplified as positive control. Arrows indicate positions of bands based on cDNA (in case of ef1α, the size of cDNA and genomic DNA is identical). Undiluted first-strand cDNA was amplified with 35 cycles, except for ef1α cDNA, which was amplified from 1:10 diluted first-strand cDNA. The multiple bands obtained with BC1G_04521-specific primers might be due to different splicing variants. The weak bands indicating the presence of wild type bhp3 genomic DNA in the triple hydrophobin mutant seem to result from the presence of few remaining, non-transformed nuclei. B: Results of real-time RT-PCR, showing gene expression in conidia and selected growth stages of strain B05.10, except for fruiting bodies which were from a cross of B. cinerea field isolates. Hydrophobin expression levels are shown relative to the mean of actin and ef1α expression.