Characterization of the aodA, dnmA, mnSOD and pimA genes in Aspergillus nidulans

Abstract

Mitochondria play key roles in cellular energy generation and lifespan of most eukaryotes. To understand the functions of four nuclear-encoded genes predicted to be related to the maintenance of mitochondrial morphology and function in Aspergillus nidulans, systematic characterization was carried out. The deletion and overexpression mutants of aodA, dnmA, mnSOD and pimA encoding alternative oxidase, dynamin related protein, manganese superoxide dismutase and Lon protease, respectively, were generated and examined for their growth, stress tolerances, respiration, autolysis, cell death, sterigmatocystin production, hyphal morphology and size, and mitochondrial superoxide production as well as development. Overall, genetic manipulation of these genes had less effect on cellular physiology and ageing in A. nidulans than that of their homologs in another fungus Podospora anserina with a well-characterized senescence. The observed interspecial phenotypic differences can be explained by the dissimilar intrinsic stabilities of the mitochondrial genomes in A. nidulans and P. anserina. Furthermore, the marginally altered phenotypes observed in A. nidulans mutants indicate the presence of effective compensatory mechanisms for the complex networks of mitochondrial defense and quality control. Importantly, these findings can be useful for developing novel platforms for heterologous protein production, or on new biocontrol and bioremediation technologies based on Aspergillus species.

Figure 1. Comparison of the growth, stress sensitivities, cell death and autolysis of the control and mutant strains.

Part A: Growth of control (THS30.3) and mutant strains on MNM agar plates. Part B: Selected stress sensitivity experiments with the control and the ΔmnSOD, ΔpimA and pimAOE strains. Part C: Growth inhibiting effect of the apoptosis-inducing antifungal protein PAF on the ΔmnSOD mutant in comparison to the control strain. Part D: Proteinase activities as autolysis markers recorded in carbon-starving (24 h) submerged cultures of the THS30.3 control and ΔaodA and aodAOE mutants. In proteinase assays, azocasein was used as substrate, and mean ± SD proteinase activity values calculated from three independent experiments are presented. The statistically significant difference calculated for the ΔaodA strain by the Student’s t test is marked with asterisks: ***p < 0.1%. Further phenotypes were not detected as shown in Supplementary Figs S1 and S3.

Figure 2. Respiration rates.

Changes in total (■), KCN-sensitive () alternative oxidase (AOX; ) and KCN + SHAM-resistant respirations () ( μmol/min/g DCM) recorded either in young cultures supplemented with 2% glucose (Part A) or in old, carbon starving cultures (Part B). KCN-sensitive and AOX-dependent respiration were calculated by substraction of KCN-resistant from total respiration and of KCN-SHAM from KCN respiration, respectively. In Part A, the addition of KCN even stimulated the respiration of the aodAOE strain and, hence, we got a virtual negative value for the KCN-sensitive respiration in this case. This paradoxical behavior can be explained with the considerably increased capacity of the AOX-dependent respiratory pathway similar to that observed before in the P. anserina AOX overexpression strain by Lorin et al.. Mean ± SD values calculated from three independent experiments are presented. Statistically significant differences compared to the control determined by the Student’s t test are marked with asterisks: *p < 5%, **p < 1%, ***p < 0.1%. In all respiration rate assays, the THS30.3 strain was used as the control strain.

Figure 3. Visualization and characterization of mitochondria.

Panel A: Three-channel confocal Z-stack images were taken where the green channel shows mitochondria (Mt) stained by MitoTracker Green (a), the red channel visualizes the superoxide (O₂^∙−) indicator dihydroethidium (b), while the blue channel (chitin staining by Calcofluor White) shows structure of the hypha and the boundaries for each segment (c). Panel B: Visualizing mitochondria-identifying these cell organelles. (d) Mitochondria stained by MitoTracker Green. (e) 2nd wavelet level of the 2D SWT of the same frame. (f) Contours of ROI (solid yellow line) and background (dashed yellow line) selected manually and mitochondria (solid white line) as provided by the automatic segmentation. (g) Identified mitochondria (cyan colored areas) superimposed to the denoised image. Panel C: Comparing mitochondrial morphology in the second hyphal segment of the THS30.3 control strain (h–j), ΔdnmA (k–m) and pimAOE strains (n–p). Markings on subpanels (i,l and o) correspond to those on subpanel (f). Markings on subpanels (j,m and p) correspond to those on subpanel (g). Scale bar: 10 μm. Panels D and E: Comparison of the volumetric ratio and size of mitochondria in the THS30.3 control, and the ΔdnmA and pimAOE mutant strains. Mean ± SD values calculated from four independent experiments are presented. Statistically significant differences determined by the Student’s t test are marked with asterisks: **p < 1%, ***p < 0.1%. No further phenotypes concerning mitochondrial morphology were observed (Supplementary Fig. S4).

Figure 4. Cleistothecia and conidiospore productions and viability of conidia.

Part A: Cleistothecia productions observed after 5 (■) and 8 d () incubations. Both the deletion and overexpression of aodA stimulated meanwhile the overexpression of mnSOD and of pimA hindered the formation of fruiting bodies. Photographs show 8 d cleistothecia of the control (THS30.3) and ΔpimA strains. Note that cleistothecia of the ΔpimA strain were sterile. (Scale bars: 200 μm). Part B: Conidiospore productions. All gene deletion strains and the pimAOE overexpression strain produced less conidiospores than the control strain. Part C: Heat stress sensitivity of the ΔmnSOD conidia. Conidia without heat treatment (50 °C for 10 min) were used as control. As shown in Supplementary Fig. S5, only conidia of the ΔmnSOD strain were sensitive to heat stress. Part D: Decreasing asexual spore viabilities during storage at 4 °C. Symbols represent the following strains: , THS30.3 (control strain), , ∆aodA, , ∆dnmA, , ∆mnSOD and, , ∆pimA. The spore viabilities of all gene deletion strains were significantly lower (p < 5% at 6 and 12 d incubation times) than that of the THS30.3 control strain. Note that no significant decreases in the conidiospore viabilities were recorded in the gene overexpression strains and, hence, these data are not shown here for clarity. In Parts A-C, mean ± SD values calculated from three independent experiments are presented. Significant differences determined by the Student’s t test are marked with asterisks: **p < 1%, ***p < 0.1%. In Part D, per cent decreases in the spore viabilities (mean ± SD values calculated from three independent experiments) are presented.

Figure 5. Summary of functions of AodA, DnmA, MnSOD and PimA in A. nidulans.

Major functions revealed by the modulation of mitochondrial morphology and functions via the deletion and overexpression of the genes aodA, dnmA, mnSOD and pimA are summarized. Schematic mitochondrial, hyphal, cleistothecial and conidiophore structures are shown. In the dividing mitochondria, hypothetical protein structures and shapes are presented, which are based on homologous proteins characterized in various species (© Ibolya Pócsi; for further details see Supplementary Note S1).