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Aspergillus fumigatus RasA Regulates Asexual Development and Cell Wall Integrity

ABSTRACT The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The ΔrasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The ΔrasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The ΔrasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a variety of diseases, ranging from allergic reactions to invasive pulmonary infection. The pathogenicity of this organism has been attributed to many factors, including thermotolerance, rapid growth of hyphae, and small conidial size (16). The conidia of A. fumigatus, considered the infective propagule, are ubiquitous in nature and are inhaled and cleared by immunocompetent hosts. However, in immunosuppressed patients, inhaled conidia survive and initiate growth. After germination and apical extension, the organism penetrates the pulmonary epithelium and vascular endothelium. In the vessel lumen, shear forces sever portions of the hyphae and deposit fungus-laden emboli in areas rich in microvasculature, leading to dissemination of the infection. Once dissemination has occurred, the mortality rate ranges from 30 to 85%, depending on the patient population (20).
The ability of the fungus to sense and respond to external stimuli is vital to the growth of the organism. Recently, considerable effort has been directed toward deciphering the signal transduction molecules involved in receiving and transmitting these external stimuli (19). The Ras superfamily, a large group of monomeric GTPase proteins, represents an important subset of these signaling molecules. Of the known Ras subfamily homologs, most act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. Among the yeasts, Ras genes play roles in normal growth, and in some cases virulence, by contributing to a wide variety of cellular processes (reviewed in reference 41). Ras1p and Ras2p are the prototypical Ras proteins of Saccharomyces cerevisiae. The importance of RAS2 in environmental sensing was first identified by its role in growth on nonfermentable carbon sources (13). Ras2p is also necessary for entry into the cell cycle during S. cerevisiae germination and, in combination with the Ras proteins Ras1p and Rsr1p, is required for completion of mitosis (12,29). Deletion of RAS2 in S. cerevisiae leads to slow growth rates, decreased intracellular cyclic AMP (cAMP) levels, accumulation of storage carbohydrates, and an increase in heat shock resistance, whereas expression of an activated form of Ras2p leads to hypersensitivity to stress, failure to arrest at G 1 upon nutrient limitation, and loss of viability at the stationary phase (14,23,37). The genome of a second model yeast, Schizosaccharomyces pombe, encodes only one Ras gene, ras1. S. pombe ras1 is required for mating, since its deletion leads to a sterile phenotype (9). Strains lacking ras1 also have abnormal cell shape and do not undergo polarized growth.
Ras homologs have been implicated in the growth and virulence of several yeast pathogens. In the human pathogen Cryptococcus neoformans, Ras1p was shown to be important for high temperature growth and, therefore, virulence (1). Deletion of ras1 also leads to defects in mating and haploid filamentous growth. The contribution of CaRAS1 to morphology and virulence has been studied in Candida albicans. CaRAS1 deletion mutants are viable and capable of forming pseudohyphae but fail to form germ tubes and hyphae in the presence of serum (6). When a dominant active allele of CaRAS1 is expressed, germination and hyphal growth are in-duced in the absence of serum (6). Since CaRAS1 helps to regulate the dimorphic switch from yeast to hyphae, a phenotype closely linked to virulence, it is not surprising that the CaRAS1-null strain has been shown to be avirulent in a mouse model. Finally, Ras activity has also been studied in the plant pathogen, Ustilago maydis. Deletion of U. maydis ras2 results in a reduction in filamentous growth, leading to a rounded-cell phenotype (18). These cells, due to lack of filamentous growth, are nonpathogenic.
To date, the functions of Ras among the filamentous fungi have been limited to the study of dominant-active and dominant-negative mutants, normally expressed in a wild-type (wt) Ras background. Isolation of a viable deletion mutant in homologs of RasA has never been reported among the filamentous fungi, suggesting either that RasA activity is essential or that deletion leads to such severe growth inhibition that isolation and characterization is impractical. Although a rasA deletion mutant of Aspergillus nidulans has never been isolated, high-level expression of a dominant-active RasA leads to precocious germination and inhibits asexual development (25,34). Similar results were seen with expression of dominant-active RasA in A. fumigatus (8). Expression of either a dominantactive or dominant-negative Penicillium marneffei rasA allele results in a delay in germination and abnormal isotropic growth (2). In addition, expression of an activated ct-Ras allele in the plant pathogen, Colletotrichum trifolii, affects polarized growth and causes reduced conidiation, whereas dominantnegative ct-Ras expression decreases germination and growth rates (38). These data, taken together, indicate that RasA homologs of the filamentous fungi play important, conserved roles in germination and the polarized growth of hyphae.
We report here the first successful isolation and characterization of a rasA deletion mutant in a filamentous fungus. Unlike the rasA homologs found in A. nidulans, P. marneffei, and C. trifolii, the rasA gene of A. fumigatus was nonessential, although deletion led to severe growth defects characterized by severely malformed hyphal and colony morphologies and nuclear aberrancies. In addition, a surprising phenotype of increased sensitivity to cell wall stress was identified in the ⌬rasA mutant. These data demonstrate that RasA is necessary for normal germination and development in A. fumigatus and provide evidence that RasA contributes to cell wall integrity.
A. fumigatus strains and growth conditions. A. fumigatus strain H237 is a clinical isolate and served as the wt strain for these studies. Strain H237, the ⌬rasA mutant strain, and the ⌬rasAϩrasA reconstituted strain (described below) were maintained on and harvested from Aspergillus minimal medium (AMM) agar plates, modified to contain 10 mM ammonium tartrate as a nitrogen source, and no supplement solution (3). Submerged cultures for determination of biomass and germination rates were grown in AMM broth as indicated. Germination experiments were performed by inoculating AMM broth with 10 5 conidia/ ml, followed by incubation over coverslips at 37°C. At the indicated times, coverslips were removed from the culture, and the number of conidia with visible germ tubes were counted. To visualize morphological changes in response to cell wall stress, AMM broth was inoculated with 10 5 conidia/ml and incubated on coverslips for 16 h with the indicated concentrations of cell wall stress agents. At the end of 16 h, coverslips were removed and mounted for analysis using an Olympus BH-2 microscope. Determination of total biomass in liquid culture was performed by inoculating 10 4 conidia into preweighed, sterile test tubes containing 5 ml of AMM broth, followed by incubation at 37°C with shaking at 250 rpm. After 24 and 48 h of incubation, the tubes were frozen in an ethanol-dry ice bath and then lyophilized for 24 h before a final weight was recorded. For growth rate analysis on solid medium, 10 4 conidia were spotted in the center of AMM agar plates, followed by incubation at specified temperatures. The change in colony diameter was recorded every 24 h for up to 5 days. Growth rates were based on the change in diameter (in millimeters) between 24 and 48 h. All of these experiments were performed in triplicate. For the determination of conidial yield on osmotically stabilized medium, 100 l of 10 5 conidia/ml were spread onto AMM agar plates with or without the indicated osmotic stabilizer. After 5 days of incubation at 37°C, 5 ml of sterile water was poured over the plate, and conidia were harvested by using a sterile swab. Harvested conidia from each strain were then filtered and counted on a hemacytometer.
In order to compare the microscopic morphologies of the conidiophores, cultures of the members of the rasA isogenic set were grown up on AMM, osmotically stabilized AMM (OSM), yeast glucose, and osmotically stabilized yeast glucose agars. Media were osmotically stabilized by the addition of 1.2 M sorbitol. The morphology of mature conidiophores, defined as those bearing conidia, was examined at 24 and 48 h for the wt and complemented strains and after 72 and 96 h for the mutant, because of the delay in germination. rasA deletion and reconstitution constructs. A deletion of the entire rasA coding region was achieved by use of double homologous recombination after transformation of protoplasts, as previously described (8). First, the primers rasA L arm-F and rasA L arm-R were designed to amplify a 3-kb region upstream of the predicted rasA translation start site. Second, the primers rasA R arm-F and rasA R arm-R were used to amplify a 2-kb region downstream of the predicted rasA stop codon. All primer sequences are provided in Table 1. These PCR fragments were then separately cloned into pGEM-T Easy (Promega) and confirmed by sequencing (University of Cincinnati DNA Core Facility). A 1.8-kb NdeI/SacI fragment of the downstream arm was then cloned into the NdeI/SacI sites of the vector containing the upstream arm, forming a new construct that contained the left flank directly connected to the right flank. To complete the construct, a hygromycin resistance cassette was then subcloned from pAN7-1 as an XhoI/XbaI fragment into SpeI/SalI restriction sites that existed at the junction of the left and right flanking regions (30). The nonlinearized deletion construct was then transformed into A. fumigatus protoplasts and plated under hygromycin  Hygromycin-resistant colonies were isolated to new media as they arose.
The ⌬rasA mutation was reconstituted by incorporation of a single copy of wt rasA locus adjacent to the deleted locus to make the reconstituted strain called the ⌬rasAϩrasA strain. The plasmid was built as follows. The primers rasA Comp-F and rasA Comp-R were used to PCR amplify a 2-kb region containing the rasA locus plus 1 kb of the 5Ј flank from the A. fumigatus genome. This PCR fragment was cloned in PCR-TOPO 2.1 (Invitrogen) and sequenced (University of Cincinnati DNA Core Facility). To finalize the construct, this rasA fragment was excised from PCR-TOPO 2.1 as a BamHI-NotI fragment and cloned into the BglII-NotI restriction sites of pTPP. Vector pTPP was constructed by subcloning the phleomycin resistance cassette from pBCphleo (Fungal Genetics Stock Center) as a SalI-HindIII fragment into pSL1180 (Pharmacia) digested with XhoI-HindIII. To complete the pTPP vector, the trpC terminator from pAN7-1 was subcloned as a SacII-SacI fragment, upstream of the newly cloned phleomycin resistance cassette. The addition of the trpC terminator sequence is to act as a terminator sequence for any gene cloned into pTPP. The final rasA reconstitution construct was then transformed into ⌬rasA protoplasts and plated under phleomycin selection (125 g/ml). Plates were incubated at 30°C and monitored for 1 week. Phleomycin-resistant colonies were isolated to new media as they arose.
Southern blot and RT-PCR analyses. DNA and RNA were extracted as previously described (8). In order to confirm that rasA deletion and reconstitution was achieved, Southern blots were performed by using EcoRI to digest the genomic DNA. Blots were probed with a fragment of the rasA promoter sequence, bp Ϫ500 to Ϫ1 from the rasA translation start site. A graphical representation of this deletion and reconstitution scheme is provided in Fig. 1. For reverse transcription-PCR (RT-PCR) analysis of the rasA transcript levels, mRNA was isolated from the isogenic set by using the mRNA Catcher PluS 96-well plate (Invitrogen). Isolated mRNA was quantitated, and equal amounts were used for PCR amplification. PCR was carried out using the rasA-specific primers rasA RT-PCR-F and rasA RT-PCR-R.
Relative expression levels by RT-PCR. Hyphae from each member of the isogenic set were harvested from AMM after 21 h of incubation at 37°C and 200 rpm and used as the starting material for RNA isolation (RNeasy; Qiagen). RT-PCR to determine transcript levels of fksA, modA, mpkA, sakA, pkaC1, pkaC2, and pkaR was performed on first-strand cDNA (Superscript III; Invitrogen) made from the extracted RNA after treatment with DNase (RQ1 DNase; Qiagen). The Sybr green-stained band intensity was normalized to gpdA, as previously described (27). Mean ratio values were determined from three independent experiments, and analysis of variance was used to analyze differences among the members of the isogenic set for a given experimental gene. Controls in which reverse transcriptase was omitted were performed with each primersample combination. All of the primers are listed in Table 1.
Staining of nuclei and the cell wall. Staining of nuclei, septa, and cell walls was performed by a modification of previously described protocols (21,26). In short, freshly harvested conidia were incubated on coverslips immersed in AMM broth. At specified times, coverslips were removed, washed twice in 50 mM PIPES (pH 6.7) and fixed in 3 ml of fixative solution (8% formaldehyde in 50 mM PIPES [pH 6.7], 25 mM EGTA [pH 7.0], 5 mM MgSO 4 , and 5% DMSO) for 30 min. After fixation, the coverslips were washed twice for 10 min in 50 mM PIPES (pH 6.7), RNase (0.1 mg/ml) was added, and the samples were incubated at 37°C for 1 h. The coverslips were then washed twice for 10 min in 50 mM PIPES (pH 6.7) and inverted onto 0.5 ml of freshly made staining solution (propidium iodide [12.5 g/ml; Sigma] and fluorescent brightener 28 [0.4 g/ml; Sigma] in 50 mM PIPES [pH 6.7]) for 5 min at room temperature. Finally, the coverslips were washed twice for 10 min in 50 mM PIPES (pH 6.7) and mounted in 50% glycerol. Fluorescence microscopy was performed on an Olympus AX80 microscope using a digital camera equipped with a DAPI (4Ј,6Ј-diamidino-2-phenylindole)/fluorescein isothiocyanate/TRITC (tetramethyl rhodamine isothiocyanate) triple cube filter. Digital images were analyzed using Spot Advanced and Photoshop 7.0 software. Bright-field and differential interference contrast analysis were performed on an Olympus BH-2 microscope.
CMFDA fungal growth assay. The chloromethyl-fluorescein diacetate (CMFDA) assay for fungal growth was performed, with slight modification, as previously described (44). Into a masked, 96-well optical bottom plate (Nunc), 10 5 conidia were added per well in 50 l of AMM. The final assay volume of 100 l was made up with 50 l of AMM containing the various cell wall stress agents at a 2ϫ the assay concentration. Control wells contained various concentrations of cell wall stress agents with no conidial inoculum. After 16 to 18 h of incubation at 37°C, 100 l of 5 M CMFDA in a buffer containing 110 mM glucose, 10 mM NaCl, and 10 mM HEPES (pH 7.2) was added to each well, and the plate was incubated as before for 1 h. Fluorescence was measured by using a FLUOstar Optima plate reader with a 485-nm excitation filter and a 520-nm emission filter. Each strain and treatment was loaded in triplicate for each experiment, and all experiments were performed three times. The results are presented as a percentage of CMFDA fluorescence calculated by comparison of total fluorescence of treated wells versus untreated wells.
Growth assays with pharmacologic agents. Plates of AMM containing graded concentrations of cyclosporine or the DMSO vehicle were inoculated with 10 4 conidia harvested from each member of the rasA isogenic set. Plates were incubated at 37°C for 72 h.
Conidia (10 4 ) of each member of the isogenic set were inoculated into wells in a 24-well plate containing AMM and OSM. One series of wells contained 10 mM

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FORTWENDEL ET AL. EUKARYOT. CELL cAMP, and a second series contained 10 mM dibutyryl cAMP. The plates were incubated for 48 h at 37°C. Cell wall assays for ␤-glucan. Flasks of AMM were inoculated with 10 5 conidia per ml and incubated at 37°C and 200 rpm for 18 to 21 h. Hyphae were harvested by centrifugation, washed, and processed for ␤-glucan content by using a modification of an aniline blue dye procedure (15,33). Approximately 5 mg of lyophilized hyphae was used as the starting material. Yeast glucan was used as the standard. The fluorescence was read on a FLUOstar Optima, using an excitation wavelength of 405 nm and an emission wavelength of 520 nm.

Deletion of A. fumigatus rasA.
Previous studies in filamentous fungi used dominant-active or dominant-negative alleles of RasA to study RasA function. In the present study we sought to gain insight into RasA function by replacing the entire rasA coding region with a dominant selectable marker encoding hygromycin resistance (Fig. 1A). In order to ensure that the ⌬rasA mutant phenotypes were the result of rasA deletion alone, a rasA complementation construct was introduced via a single crossover integration in the rasA promoter region (Fig. 1A). Deletion and reconstitution of the rasA coding sequence was verified by Southern blot analysis (Fig. 1B). Genomic DNA from each strain of the isogenic set was digested with EcoRI and hybridized with a 32 P-labeled probe from the rasA promoter sequence. The wt and deletion strains yielded a ϳ6-kb and a ϳ4-kb band, respectively, and the complemented strain (⌬rasAϩrasA) produced a 3-kb band corresponding to the original deletion locus and a 10-kb band from integration of the complementation construct. These band sizes were as predicted based on the introduction and/or deletion of EcoRI restriction sites within the region. As further confirmation, RT-PCR using gene-specific primers confirmed the loss, and reappearance, of rasA mRNA in the ⌬rasA and ⌬rasAϩrasA strains, respectively (Fig. 1C).
Loss of RasA causes severe growth inhibition. When grown on solid media, the ⌬rasA mutant formed a tightly compact colony with limited capacity for radial outgrowth, accompanied by nearly absent conidiation ( Fig. 2A and Fig. 3). The diameter of the ⌬rasA mutant colony was only ca. 50% of the size of the wt colony after the first 48 h of growth at 30 or 37°C. No further substantial outgrowth was noted for the mutant over the next 72 h of incubation at 37°C. When the coding region of rasA was reinserted into the genome of the deletion mutant, both the growth and conidiation defects were complemented. Because this severe reduction in colony diameter is similar to the phenotype of the calcineurin catalytic subunit deletion mutant, the sensitivity of ⌬rasA to the calcineurin inhibitor cyclosporine was examined (Fig. 3). The ability of the mutant to respond to cyclosporine suggested that the calcineurin pathway was still intact in the ⌬rasA mutant (4,35).
Interestingly, growth on media containing 1.2 M sorbitol remediated the reduced conidiation phenotype of the ⌬rasA strain (Table 2) but did not ameliorate the reduced ⌬rasA radial growth (Fig. 3). Although improved conidiation could be achieved by supplementation of the medium with either organic or inorganic osmotic stabilization compounds, osmotic stabilization of the medium did not prevent the appearance of abnormal conidiophore morphology, such as a "double-vesicle" head at the tip of the conidiophore (Fig. 4D). Abnormal morphology, including branched, rudimentary, or septate co-nidiophores. was detected in 30 to 35% of the ⌬rasA conidiophores isolated from osmotically stabilized media, whereas both the wt and the ⌬rasAϩrasA strains showed at most 20% abnormal (septate stalks only) conidiophores on the same medium (Fig. 4). Even in the most normal-appearing mutant conidiophores, the phialides were notably blunted or rounded at the tip, rather than being the normal, "bowling pin" shape, as seen in the wt and complemented strains.
Exogenous cAMP does not complement the ⌬rasA growth defect. Because some of the morphological and developmental phenotypes seen in C. albicans and C. neoformans can be remediated by the addition of exogenous cAMP, the growth of the rasA isogenic set was compared on AMM with or without 10 mM cAMP or dibutyryl-cAMP (1,17,24). Neither of the cyclic nucleotides, either tested alone or in combination with an osmotic stabilizer, was able to restore wt phenotypic features to the rasA deletion mutant (Fig. 3 and data not shown).
Deletion of rasA impairs germination and hyphal development. In medium containing glucose as the sole carbon source, the ⌬rasA mutant displayed decreased germination rates, requiring nearly 3 h longer than wt or ⌬rasAϩrasA strain to reach 100% germination (Fig. 2B). However, ⌬rasA conidia were 100% viable (data not shown). In liquid culture, the total hyphal mass of the ⌬rasA mutant was greatly decreased after 24 and 48 h of growth, even after correcting for the germination delay (Fig. 2C). No difference in biomass accumulation was noted between the wt and reconstituted strains. Microscopic examination of hyphae formed in submerged culture (Fig. 5) revealed striking morphological defects in the ⌬rasA mutant. After 16 h of submerged growth, ⌬rasA hyphae displayed a stunted phenotype with frequent changing of the axis of polarity. In contrast to wt A. fumigatus, which was able to maintain a single, constant growth axis over 50 m of growth, the ⌬rasA hyphae changed their axis of growth polarity as many as seven times (Fig. 5). Mutant hyphae were more than two times the diameter of wt hyphae and appeared more refractile under light microscopy. These abnormal hyphal development characteristics were not seen in the wt or reconstituted strains under similar conditions. RasA regulates nuclear morphology and distribution. Because alteration of RasA activity has been shown to cause aberrant mitotic events in both A. fumigatus and A. nidulans, nuclear division and distribution was examined in the ⌬rasA mutant (8,34). After 16 h of culture in YG medium, wt hyphae, stained with propidium iodide and calcofluor white, displayed an average of five to seven evenly spaced nuclei per subapical compartment (Fig. 6). These data are in keeping with the previously reported averages for A. fumigatus (21). In contrast, hyphae from the ⌬rasA strain contained many variably sized nuclei, unevenly distributed throughout the hyphae (Fig. 6).
Growth inhibition due to RasA deletion is not enhanced at elevated temperatures. In C. neoformans, growth inhibition of the RAS1 deletion strain worsens upon incubation at 37°C, and the mutant fails to grow at 39°C, implying that the effects of deletion and high temperature are additive (1). To test for this phenotype in the A. fumigatus rasA mutant, conidia from the wt, ⌬rasA, and ⌬rasAϩrasA strains were inoculated onto AMM and allowed to grow for 48 h at 30 or 37°C. The ⌬rasA mutant developed a colony diameter roughly 50% that of the wt and  Conidia (10 4 ) from each strain were inoculated onto coverslips and submerged in YG media at 37°C. The percent germination was scored as the number of conidia producing a visible germ tube among 100 total enumerated conidia. (C) Comparison of total biomass from the isogenic set. Each strain was grown for 24 or 48 h at 37°C in YG, and the total biomass was determined as described in Materials and Methods. All experiments were performed in triplicate. Measurements and error bars represent the mean Ϯ the standard deviation (SD).
Loss of rasA increases resistance to digestion and sensitivity to cell wall and membrane stress. Cell wall-degrading enzymes can be used to liberate A. fumigatus protoplasts from germling cells. However, several preparations of cell wall-degrading enzymes that are typically used to protoplast A. fumigatus hyphae were not effective with the ⌬rasA strain (Table 3). For example, use of a mixture of driselase, lyticase, and ␤-D-glucanase yielded no protoplasts from the ⌬rasA strain (26). In fact, even at concentrations of protoplasting enzymes that were double those effective with the wt, no protoplast formation was seen with the ⌬rasA mutant. Vinoflow FCE, when used as described for protoplast generation with Aspergillus, resulted in extremely low protoplast yield (http://www.fgsc.net/Aspergillus /Vinoflow%20protoplasting.pdf). Adequate generation of protoplasts from the ⌬rasA deletion mutant was only achieved by digesting germlings with the highly active cell wall-degrading enzyme preparation Novozyme 234. Because these data suggest differences in the cell wall composition and/or architecture of the ⌬rasA mutant compared to the wt, we next measured the ␤-glucan content in the cell walls and the steady-state message levels of fksA, the ␤-glucan synthetase. No significant differences in glucan concentration were detected among the members of the rasA isogenic set, with all three isolates averaging between 2.8 and 3.9 g of glucan per mg of dry weight. Likewise, message levels for fksA among the members of the isogenic set were not significantly different (see Fig. 9).
Since the ⌬rasA mutant did not show quantitative differences in glucan content, the sensitivity to agents known to perturb the cell wall stability of fungi, manifested as the inhibition of growth and/or abnormal morphology, was tested. When incubated at 37°C in the presence of 75 g of Congo red/ml, the growth of the ⌬rasA mutant was severely inhibited, forming large swollen cells with no polarized growth axis (Fig.  7). Similar aberrant morphology was noted in the presence of 1 g of Nikkomycin Z/ml; the ⌬rasA mutant formed large, swollen "ballooned" cells ( Fig. 7). At each of these concentrations, the wt and ⌬rasAϩrasA strains displayed minimal growth abnormalities, consisting of swollen cells interspersed with normal hyphal strands. Interestingly, aberrant phenotypes produced in the ⌬rasA mutant by treatment with each stressor could be remediated, at least partially, by the addition of 1.2 M sorbitol to the growth medium (Fig. 7). In the presence of increasing amounts of SDS, the ⌬rasA mutant displayed a sharp decrease in growth, as measured by the CMFDA fungal growth assay. However, the wt and ⌬rasAϩrasA strain were able to resist slightly higher levels of SDS ( Fig. 8A and data not shown). A similar pattern was obtained after treatment with caffeine and Fungin. The ⌬rasA mutant displayed high sensitivity to very low levels of Fungin (0.75 g/ml) compared to the wt and complemented strains, and growth was reduced in 3 mM caffeine for ⌬rasA ( Fig. 8B and C, respectively). The inhibition of growth occurred in a concentration-dependent manner for SDS, caffeine, and Fungin alike. Despite the phenotypic differences in response to cell wall perturbation seen in the ⌬rasA mutant, transcriptional responses were not detected in mitogen-activated protein kinases (MAPKs) involved in cell wall integrity (mpkA and sakA), in the Rho GTPase involved in control of growth polarity (modA), or in protein kinase A  3. Deletion of rasA causes restricted radial growth. Colonies of the members of the rasA isogenic set were compared after 48 h of incubation at 37°C. The addition of 1.2 M sorbitol did not correct the radial growth defect (YG versus YGϩsorb), but conidiation was restored. The addition of exogenous cAMP (as dibutyryl-cAMP [Db-cAMP]) to OSM did not remediate the growth phenotype, even in the presence of sorbitol (OSMϩDbcAMP). Inhibition of the calcineurin pathway by the addition of 75 M cyclosporine (Cs) to AMM resulted in radial growth inhibition of all members of the rasA isogenic set, implying that calcineurin is active in the rasA deletion mutant.

DISCUSSION
The data provided here reveal roles for A. fumigatus RasA in many stages of the asexual growth cycle, including the development of proper cell wall integrity; the timing of germination, hyphal morphology, and growth; the nuclear distribution; and asexual development. There is ample precedent to suggest that many of these roles for Ras are conserved among the fungi. Although the molecular mechanisms by which Ras imparts this developmental control have been well studied in yeast, there are few data for the filamentous fungi. Such studies have been limited, at least in part, by the essential nature of ras genes among this group of fungal organisms. Surprisingly, the studies described here show that rasA is nonessential in A. fumigatus.
Deletion of A. fumigatus rasA led to a decrease in germination and radial growth rates and severe defects in hyphal morphology, with all phenotypes indicative of aberrancies in polarized growth. Although contributions of Ras activity to polarized growth processes have been noted in several fungi, exploration of the molecular mechanisms has been pursued primarily in yeast. For example, the conserved Ras/Cdc42/ MAPK pathway and the Ras/PKA pathway have been implicated as possible polarity regulators in S. cerevisiae, C. albicans, C. neoformans, and U. maydis (1,17,18,22). In contrast, the only mediator of Ras activity known to control polarized growth of the yeast S. pombe is the Cdc42 signaling pathway. Our data suggest that A. fumigatus, like some other filamentous fungi, is more similar to S. pombe, since growth on exogenous cAMP, even in the presence of 1.2 M sorbitol, did not remediate the ⌬rasA growth and development phenotypes (Fig. 3). This is in contrast to what has been reported in C. albicans and C. neoformans. In C. albicans, the homozygous deletion of CaRAS leads to a profound defect in the morphological transition to filamentous growth, which can be reversed by the addition of exogenous cAMP (17). Likewise, in C. neoformans, the mating defect in the ⌬ras1 strain is, at least partially, remediated by exogenous cAMP (1). The failure of cAMP to remediate the ⌬rasA phenotypes in A. fumigatus is consistent with RasA signaling through ModA (CDC42) and MAPK pathways rather than through PKA. Indeed, in A. nidulans, Ras and PKA have also been shown to control germination processes through separate mechanisms (7).
In the model yeast S. cerevisiae, Ras also signals through Cdc42 and MAPK pathways to control pseudohyphal growth, and this interaction is mediated by 14-3-3 scaffold proteins (32). In S. pombe, signaling through the Ras/Scd1 (a guanine exchange factor for Cdc42) pathway is believed to be important for actin and microtubule assembly, both of which are important for proper polarized growth (28). In fact, attempts to obtain null mutants of Cdc42 in the filamentous fungi Ashbya gossypii and P. marneffei have been unsuccessful, suggesting that such deletions are lethal (2,42). However, in filamentous   A and B) and ⌬rasA (C to E) strains grown on YG plus 1.2 M sorbitol for 2 days at 37°C are shown. Note the septated conidiophore in panel B (arrowhead), seen in 15 to 20% of wt and ⌬rasAϩrasA conidiophores on sorbitol containing medium. A minority of mutant conidiophores were relatively normal (C), but even those showed blunted phialides. Many mutant conidiophores were branched (D) or bizarre and rudimentary (E). Conidiophores from the ⌬rasAϩrasA strain (not shown) were indistinguishable from the wt. All were photographed at 400ϫ using differential interference contrast (panels A to C and panel E) or bright-field microscopy (panel D).

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FORTWENDEL ET AL. EUKARYOT. CELL fungi, as in higher eukaryotes, there is redundancy in the pathway in the form of Rac GTPases. Therefore, it is perhaps not surprising that studies in A. nidulans have illustrated redundant and overlapping roles of the two GTPases, which allows the deletion of CDC42 (modA) in this filamentous fungus (40).
Interestingly, in addition to a markedly restricted radial growth phenotype in A. nidulans after CDC42 (modA) deletion, the mutant hyphae was broader and contained more nuclei than the wt. These phenotypes are similar to what we have observed in the ⌬rasA mutant, and the increased number of nuclei may be a common response in mutants in which there is increased cytoplasmic volume and reduced hyphal extension, such as the sepA1, ⌬CDC42, and ⌬rasA mutants (11,40). The Ras/Cdc42/ MAPK pathway is likely a conserved module that functions in the regulation of hyphal growth in A. fumigatus and may be a downstream target of RasA activity. However, we saw no evidence of changes in transcript abundance in the ⌬rasA mutant compared to the wt or the complemented strains (Fig. 9). This does not rule out regulation by translational or posttranslational mechanisms, which we are actively pursuing. Deletion of A. fumigatus rasA led to an almost complete loss in conidiation that could be recovered by growth on osmotically stabilized medium (Table 3 and Fig. 3). Even though nearly wt levels of conidia were produced, microscopic observations of the conidiophores revealed that morphological abnormalities remained. The rudimentary conidiophores predominated, but even those that looked relatively normal showed blunted phialides and often were septate. The septated conidiophore stalks appeared to be stimulated in hyperosmotic medium, however, based on their increased frequency in the wt and ⌬rasAϩrasA strains (Fig. 4). Similar phenotypes have been reported in A. nidulans, in which expression of alleles carrying dominant-active or dominant-negative mutations of RasA leads to aberrant conidiophore morphology or cessation of the asexual developmental program, respectively (34). Another commonly reported phenotype of Ras mutants in fungi is the increased sensitivity to heat shock and/or high-temperature growth. Strains of S. cerevisiae and C. albicans expressing activated Ras alleles are hypersensitive to heat shock, whereas a C. neoformans Ras1 deletion strain displays reduced growth at elevated temperatures (1,6,13). Interestingly, the reduced growth phenotypes seen in the A. fumigatus rasA deletion mutant or in a strain expressing an activated allele were not related to the incubation temperature, at least under the conditions we examined. Perhaps this is because A. fumigatus, in contrast to the other fungi mentioned, is known to be thermotolerant, showing an optimal growth temperature of approxi-  7. Treatment of the rasA mutant with Congo red or Nikkomycin Z results in morphologically abnormal growth. Conidia (10 5 ) from the wt and ⌬rasA strains were grown in AMM with the addition of 75 g of Congo red or 1 g of Nikkomycin Z/ml overnight at 37°C. Images are shown from cultures with (w/) or without (w/o) 1.2 M sorbitol (Sorb) added to the medium as an osmotic stabilizer. mately 42°C. However, when taken together, these data indicate that many, but not all, of the reported rasA mutant phenotypes are conserved among the fungi. The most surprising of the ⌬rasA phenotypes revealed in the present study is the increased sensitivity noted when the mutant is grown in the presence of cell wall stressors: caffeine, used to test cell wall integrity in yeast; Congo red, an inhibitor of cell wall microfibril assembly; Nikkomycin Z, a chitin-synthase inhibitor; SDS, a detergent that disrupts the cell membrane; and Fungin, a polyene that disrupts ion exchange through the cell membrane. That the mutant might have abnormalities associated with its cell wall was first suggested by the difficulty in making protoplasts from the mutant when we were attempting to complement the mutation. The preparations used for protoplasting in Aspergillus are complex mixtures of various enzymes; most are culture filtrates, the components of which are not described in detail (Novozym 234) or are proprietary in nature (Vinoflow FCE). The combination of ␤-glucanase [which attacks the ␤-(1-4)-D-glucan linkages], lyticase [which hydrolyzes the ␤-(1-3)-D-glycopyranosyl linkages], and driselase (a crude powder containing laminarinase, xylanase, and cellulose) was not effective in digesting the cell walls from the ⌬rasA mutant. Likewise, Vinoflow FCE, a proprietary mixture from Novozymes that is marketed as a wine maturation agent, was ineffective in making protoplasts. Only Novozym 234, a culture filtrate from Trichoderma harzianum reported to contain ␣-(1-3)-glucan glucanohydrolase and ␤-(1-3)-glucan glucanohydrolase, among other components, was able to make protoplasts readily from the ⌬rasA mutant (5,31). Because of the difficulty in making direct comparisons, either qualitative or quantitative, among the different protoplasting mixtures, we attempted to determine whether there were recognizable differences in cell wall composition or integrity that would help to explain our findings.
The cell wall integrity of the ⌬rasA mutant was compared to the wt and reconstituted strains under stress induced by exposure to a known cell wall intercalating agent, a chitin synthase inhibitor, and cell membrane disruptors. The ⌬rasA strain displayed decreased growth and/or abnormal morphology in the presence of all of the cell wall agents tested. In an effort to understand whether these phenotypes might be mechanistically related, we measured the ␤-glucan content of the cell walls, the transcript abundance of fksA, the ␤-glucan synthetase gene, and the integrity of the calcineurin signaling pathway, reasoning that that loss of cell wall integrity might be due to decreased structural ␤-glucan. Using aniline blue fluorescence, we were unable to detect any differences in ␤-glucan content among the members of the rasA isogenic set. Although the transcript level was somewhat elevated, rather than decreased as we had hypothesized, in the ⌬rasA mutant, this increase was not significant. The calcineurin signaling protein, CnaA, has been shown to regulate ␤-glucan synthesis in A. fumigatus, and deletion of rasA results in phenotypes similar to those seen in the ⌬cnaA mutant, including increased sensitivity to Nikkomycin Z (36). However, calcineurin signaling was still intact in the ⌬rasA mutant, as displayed by continued sensitivity to cyclosporine (Fig. 3). These data indicate that the difference in susceptibility to cell wall perturbation in the mutant was not due to defective ␤-glucan biosynthesis and that RasA and CnaA may mediate their effects on cell wall integrity through distinct mechanisms.
Our second hypothesis was that RasA was upstream of MpkA, the cell wall integrity MAPK in A. fumigatus or, because of the effect of sorbitol on conidiation, of SakA, the Hog1 homolog (39,43). Again, no significant differences in transcriptional abundance were detected (Fig. 9). Finally, since PKA signaling is involved in the general stress response in S. cerevisiae and in oxidative stress in A. fumigatus, the transcript levels of the genes encoding the PKA holoenzymes were examined and found to be equal in the isogenic set (10,45). Therefore, although the cell wall integrity of the ⌬rasA mutant was compromised, none of the pathways we investigated were involved via transcriptional mechanisms. However, this does not preclude regulation of the cell wall through other mechanisms.
In conclusion, the isolation of a complete rasA deletion mutant in A. fumigatus reported here is the first time such a deletion strategy has been successfully reported in a filamentous fungus. Deletion of rasA yielded some phenotypes that would be predicted, based on conserved Ras function among the fungi. These include decreased germination and growth rates, severely malformed hyphae with polarized growth defects, loss of conidiation, and abnormalities in nuclear distribution. Surprisingly, the ⌬rasA strain demonstrated an intriguing phenotype of cell wall instability. This finding opens the exciting possibility of altering RasA activity, possibly through the use of farnesyl-transferase inhibitors, in combination with cell wall antifungals as a means of reducing Aspergillus viability. Future studies with the ⌬rasA mutant will focus on defining the structure of the ⌬rasA cell wall and delineating the pathways in which RasA acts to regulate asexual development and cell wall integrity.
We thank Jay Card for his assistance with photography and figures.
FIG. 9. Transcript abundance in members of the rasA isogenic set compared using RT-PCR. RNA from wt (Ⅺ), ⌬rasA (f), and ⌬rasAϩrasA (u) was harvested from hyphae grown overnight as described in Materials and Methods. Products of the RT-PCR were run on gels and stained with Sybr green, and the intensity in pixels was normalized to the signal from gpdA run in the same gel. These ratios were then analyzed by analysis of variance, and no significant differences were detected.