Figure 1.
DCB decreases growth of A. nidulans mycelium.
Growth of A. nidulans cultivated on solid MM supplemented with different concentrations of DCB, measured by taking the diameter of the mycelium over 6 days. Inset: images showing the mycelium grown for 2 and 3 days with increasing concentrations of DCB. Bars refer to 1 cm.
Table 1.
Effect of DCB on the growth of the different A. nidulans strains used in this study and on A. niger.
Figure 2.
DCB and CR cause the formation of aberrant hyphal structures.
Bright field microscopy pictures (40x in A, B and C, and 60x in D) of mycelium grown in liquid MM (A), in medium supplemented with 100 µM CR (B), 1% v/v MetOH (C) and 200 µM DCB (D). The arrows point to aberrant structures present along the hyphae (namely swollen regions and bulges). Scale bar refers to 10 µm.
Table 2.
Effect of cell wall drugs alone or in combination with DCB on the different A. nidulans strains studied.
Figure 3.
Germination of conidiospores in the presence of 100 µM CR and 200 µM DCB.
Figure 4.
Gene expression analysis in CR and DCB-treated mycelium.
Quantitative real-time PCR analysis of cell wall biosynthetic genes in A. nidulans grown in the presence of (A) 100 µM CR and (B) 200 µM DCB for 0, 1, 3, 6 and 24 h. Asterisks indicate significant (*) and very significant (**) changes.
Figure 5.
DCB does not affect chitin content and triggers the formation of wall “ghosts”.
Confocal microscopy pictures of control (A and B) and DCB-treated A. nidulans (C and D). Differential Interference Contrast (DIC) images in (A) and (C) and CFW fluorescence images in (B) and (D). Arrows point to cell wall “ghosts”. Bars refer to 10 µm.
Table 3.
Protoplasting efficiency (in %) of control and DCB-treated A. nidulans.
Figure 6.
Hyphal ultrastructural features in the absence and presence of drugs.
SEM pictures of (A) control, (B) CR-, (C) MetOH- and (D) DCB-treated hyphae. Arrows point to aberrant structures. Scale bar refers to 20 µm.
Figure 7.
Hyphal ultrastructural differences triggered by CR and DCB.
(A–B): SEM pictures of (A) CR- and (B) DCB-treated mycelium, showing the presence of small particles accumulating on the hyphal surface. (C–L): AFM images, showing surface topographical gradient (C–F) and adhesion maps (G–L) obtained on samples grown overnight at 37°C in control condition (C and G), with 100 µM CR (D and H), with 1% v/v MetOH (E and I) and 200 µM DCB (F and L). Bars refer to 10 µm (A and B) and 2 µm (C–L).
Figure 8.
Fractal analysis of AFM images.
(A) Close-up of the adhesion maps in Fig. 6 on the hyphal surfaces. Scale bars refer to 0.5 µm. (B) Plot representing the fractal dimension D obtained from the images in (A) according to different calculation tools available in Gwyddion, namely: Drms from root mean square of adhesion values, Dcube and Dtria from differently shaped (cube or tetragonal) boxes used for coverage of the image surface, and Dspec from Fourier spectrum of the spatial frequencies contained in the image.