A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in Aspergillus fumigatus

The IRD is required for hyphal growth and asexual development. (A) Colony morphologies of the control strain, the rasA deletion mutant (ΔrasA), the IRD truncation mutant (RasAΔIRD), and mutants expressing N-terminal chimeras including the IRD regions from S. cerevisiae (ScAf), N. crassa (NcAf), and C. neoformans (CnAf). For each mutant, the N-terminal RasA protein sequence resulting from mutational analysis is displayed. Equal numbers of conidia (10⁴ total conidia) were inoculated onto the middle of AMM agar plates and allowed to grow for 5 days at 37°C. (B) Graphical representation of colony diameters from each strain (10⁴ total conidia) over 5 days of growth at 37°C. Each culture was assayed in triplicate, and results were averaged. The graph displays the average for each time point ± standard deviation. (C) Enumeration of conidia produced by each strain after 3 days of growth at 37°C. Equal numbers of conidia (2 × 10⁴ total conidia) were spread onto AMM agar and incubated for 3 days. Conidia were harvested from each plate in 20 ml of water, washed 3 times with water, resuspended in 10 ml of water, and enumerated. Data are presented as the total conidia per plate and are the average from two experiments ± standard deviation.

The invariant arginine residue is critical for IRD-mediated RasA functions. (A) Colony morphologies of the control strain, the rasA deletion mutant (ΔrasA), the alanine-scanning mutants (RasAS3A, RasAK4A, RasAF5A, RasAL6A, and RasAR7A), and the mutant with lysine mutation of the invariant arginine residue (RasAR7K). Equal numbers of conidia (10⁴ total conidia) were inoculated onto the middle of AMM agar plates and allowed to grow for 5 days at 37°C. (B) Graphical representation of colony diameters from each strain (10⁴ total conidia) over 5 days of growth at 37°C. Each culture was assayed in triplicate, and results were averaged. The graph displays the average for each time point ± standard deviation. (C) Enumeration of conidia produced by each strain after 3 days of growth at 37°C. Equal numbers of conidia (2 × 10⁴ total conidia) were spread onto AMM agar and incubated for 3 days. Conidia were harvested from each plate in 20 ml of water, washed 3 times with water, resuspended in 10 ml of water, and enumerated. Data are presented as the total conidia per plate and are the average from two experiments ± standard deviation.

IRD truncation abrogates RasA function. (A) Comparison of germination rates between the control strain and the RasAΔIRD mutant. Equal numbers of conidia were inoculated onto AMM broth and incubated at 37°C. At the noted time points, samples were removed, and 100 random conidia from each strain were scored for germ tube production (polarity establishment). Data are presented as the percentage of germinated conidia for each time point. (B) Representative micrographs of germinating conidia from each strain after 5 h of incubation at 37°C. Note the irregular conidial size of the RasAΔIRD mutant, indicating higher variability in the timing of germination initiation. (C) Representative micrographs of hyphal development aberrancies in the RasAΔIRD mutant after 16 h of growth at 37°C. Conidia from each strain were cultured as described for the germination studies. Note the stunted, highly branched hyphae produced by RasAΔIRD. (D) Representative micrographs of conidiophore development in the control strain and RasAΔIRD mutant. After 24 h of culture at 37°C on AMM agar, the control strain produced conidiophores with swollen vesicles and numerous conidia arranged in chains (left panel). The RasAΔIRD mutant produced no conidiophores at 24 h of identical culture (data not shown) and produced distorted conidiophore structures by 48 h (right panel), characterized by minimally swollen vesicles and few conidia.

RasA localization and activation are not affected by IRD truncation. (A) Representative fluorescence micrographs of the control strain and RasAΔIRD. AMM broth cultures of each strain were incubated at 37°C until approximately equal hyphal lengths were achieved for each strain. Cultures were then mounted for fluorescence microscopy, and images were acquired using identical exposure settings for both strains. (B) Ras activation assay performed on lysate from the control strain comparing the amount of total Ras bound under no treatment (NT), after GTP preloading (+GTP), and after GDP preloading (+GDP). GFP-fused RasA is detected at 51 kDa using an anti-Ras antibody. The control assay was repeated twice to ensure the ability of RasA to bind the Raf1 RBD-agarose bead and to appropriately respond to activation (+GTP) and inactivation (+GDP). Lane M, molecular mass (kilodaltons) marker lane.(C, top panel) Western blot analysis of total RasA detected in lysates from each strain (Total) and the amount of active RasA detected by Raf1-RBD pulldown (Active). The gel image is representative of three independent experiments. Ctrl, control. (C, bottom panel) Quantification of active/total Ras ratios. Western blot bands were quantified by densitometry analysis, and the average signal abundance ratio of detectable active RasA to total RasA was calculated. Data are presented as an average from three replicates ± standard deviation. No statistical difference was noted in Ras activation levels between the control and RasAΔIRD mutant.

PKA activation is deficient in the RasAΔIRD mutant. (A) PKA activity assay performed on lysates of the control strain, a strain expressing constitutively active RasA (DArasA1), and the RasAΔIRD mutant. Equal numbers of conidia from each strain were cultured in either (i) AMM glucose-based medium (Glucose 16 hr) overnight or (ii) AMM modified to contain glycerol as the sole carbon source, followed by a 2-min stimulation with glucose (Glycerol 16 hr/Glucose 2 min). Lysates were generated from each strain and condition and assayed for PKA activity using the PepTag nonradioactive cAMP-dependent protein kinase assay (Promega). The agarose gel electrophoresis image is representative of three independent experiments and shows migration of phosphorylated PKA-target peptide toward the cathode (+) and unphosphorylated target toward the anode (−). Control experiments, including addition of the bovine PKA catalytic subunit and exogenous cAMP to measure induction of PKA activity, are provided in Fig. S3 in the supplemental material. (B) Densitometric quantification of phosphorylated PKA-target peptide (migrating toward the cathode) for each strain, relative to the control strain. Results are the average from three separate experiments ± standard deviation. Data were compared using Student’s t test.

RasA interaction with Cdc42 is mediated by the IRD in A. fumigatus. (A) Lysates of the control strain and RasAΔIRD mutant were immunoprecipitated with anti-GFP agarose beads and subjected to Western blot analysis with either an anti-mCherry antibody (for mCherry-Cdc42 detection) or an anti-GFP antibody (for Ras protein detection). Images are from one representative experiment. Control experiments, including the inverse-pulldown reaction, are provided in Fig. S4 in the supplemental material. (B) Quantification of the amount of mCherry-Cdc42 immunoprecipitated with either RasA (from the control strain) or RasAIRD. Densitometry analysis was performed using ImageJ software, and the ratio of anti-mCherry signal to anti-GFP signal was calculated. The graph represents the average result from three separate experiments ± standard deviation. Data were compared using Student’s t test.

The IRD is required for RasA-mediated actin polarization. Immunofluorescence micrographs showing actin cytoskeleton distribution in the control strain (upper panel) and the RasAΔIRD mutant (lower panel). AMM broth cultures of each strain were incubated at 37°C until approximately equal hyphal lengths were achieved for each strain. Images were acquired under identical exposure times and are representative of the entire culture for both strains. Note the decreased presence of actin polarization to the hyphal tip in the RasAΔIRD mutant (inset panels). Arrowheads denote locations of hyphal tips for both panels.