nidulans. Table 2 The effect of 1 M sorbitol on the growth inhibiting activity of AFPNN5353 on A. nidulans. AFPNN5353 (μg/ml) CM CM + 1 M sorbitol 0 100 (SD ± 10) 100 (SD ± 11) 0.05 10.4 (SD ± 1) 79.3 (SD ± 6) 0.1 5.5 (SD ± 2) 68.3 (SD ± 0.8) 0.2 no growth 17.8

(SD ± 0.8) 1 × 104 conidia/ml were incubated in CM with 0-0.2 μg/ml AFPNN5353 for 24 h. Percent values were calculated from percent changes in OD620 of AFPNN5353 treated A. nidulans compared to untreated controls (= 100%). Results are expressed as mean ± SD (n = 3). To investigate whether AFPNN5353 induces agsA gene transcription PHA-848125 similar to AFP via the Pkc/Mpk Selleck Bortezomib signalling pathway, we tested the effect of the antifungal protein on the transgenic A. niger strain RD6.47 which expresses a nuclear-targeted GFP protein fused to the A. niger agsA promoter. RD6.47 germlings were treated with AFPNN5353 (conc. 10 to 100 μg/ml) for 2 h and analyzed microscopically. As shown in Additional file 1, a nuclear signal was clearly detectable in germlings of RD6.47 treated with ≥ 50 CA-4948 manufacturer μg/ml AFPNN5353, similar to that when exposed to 10 μg/ml caspofungin. In untreated germlings, however, no signal could be observed. These observations perfectly match with the data obtained for AFP [10]. It has to be noted here that antifungal protein concentrations higher than the MIC determined for conidia (> 10-50 fold) are needed

to inhibit the growth of germlings or hyphae of sensitive fungi [10, 27] (data not shown). Next, we tested several A. nidulans mutant strains affected in central players of the CWIP for their susceptibility to AFPNN5353

by determining their radial growth in the presence or absence of the antifungal protein. Since RhoA is an essential protein in A. nidulans, two strains with ectopic copies of the constitutively active rhoA G14V allele and the dominant rhoA E40I allele [28] were tested in comparison to the wild type strain (GR5). The rhoA G14V mutation prevents the hydrolysis of GTP and therefore renders RhoA constantly active [28]. Similarly, the GTP hydrolysis is inhibited in the RhoAE40I strain, but this mutation also perturbs the binding of the GTPase activating protein (GAP) to RhoA and possibly disturbs downstream effectors of RhoA-GAP [28]. The constitutively Carnitine palmitoyltransferase II active RhoAG14V and the dominant RhoAE40I strain exhibited the same sensitivity towards AFPNN5353 as the wild type strain at low protein concentrations (≤ 0.2 μg/ml) (Figure 2A). Interestingly, the dominant RhoAE40I strain was more resistant to AFPNN5353 than the wild type strain or the RhoAG14V strain at higher protein concentrations (1 μg/ml) (Figure 2A). Therefore, we suggest that the toxicity of AFPNN5353 is transmitted by RhoA-GAP targets and not by RhoA itself. These mutants performed similarly when exposed to the orthologous P. chrysogenum antifungal protein PAF [9]. Figure 2 AFP NN5353 susceptibility of A.