We isolated the ganB gene encoding the G-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.

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GUANINE nucleotide-binding proteins (G proteins) are key components in various signal transduction pathways and show great complexity in structure and function. Heterotrimeric G proteins consist of three subunits, , ß, and . The -subunit has intrinsic GTPase activity and associates with ß-subunits with bound GDP in the inactive state (GILMAN 1987; SIMON et al. 1991). When ligands interact with their seven cognate transmembrane receptors, the GDP bound to the -subunit is replaced with GTP, resulting in dissociation of the GTP-subunit from the ß-complex. The -subunit with GTP bound and the free ß-complex may interact with many types of downstream effectors such as enzymes (adenylate cyclases, phosphodiesterases, phospholipases, etc.) and ion channels, which in turn initiate signals via second messengers such as cyclic AMP, diacylglycerol, inositol triphosphate, and/or critical ions in the signal transduction pathway (NEER 1995; BOHM et al. 1997; NEVES et al. 2002). Thus signals emanating from the cell surface are delivered to diverse downstream effectors by switching G-protein functions on and off. The integrated G-protein signals are responsible for orchestrating a coherent specific biological response.

In fungi, G proteins are known to regulate various morphogenetic events and pathogenicity, including dimorphic switching, appressorium formation, asexual or sexual development, and vegetative growth (BöLKER 1998). Fungal G-proteins are classified into three major groups according to their amino acid sequences (BöLKER 1998). Groups I and III are related to the mammalian Gi- and Gs-subfamilies, respectively. Group II fungal G-proteins have no corresponding mammalian counterpart. Group III fungal G-proteins play crucial roles in growth and development, including sporulation and germination, and pathogenesis in diverse fungi (LIU and DEAN 1997; KAYS et al. 2000; LIEBMANN et al. 2003; ZUBER et al. 2003). The cAMP/PKA system is also involved downstream of group III G-protein signaling in some processes such as cell proliferation, development, stress response, mating, and virulence (KüBLER et al. 1997; KRUGER et al. 1998; ALSPAUGH et al. 2002; IVEY et al. 2002; LIEBMANN et al. 2003).

A dominant activating mutation in the fadA gene, encoding a G-subunit of the filamentous fungus Aspergillus nidulans in which the intrinsic GTPase activity has been eliminated, thereby fixing FadA in its active GTP-bound state, showed a proliferative growth phenotype and no conidiation (YU et al. 1996). This means that the active G-subunit of a heterotrimeric G protein of A. nidulans stimulates hyphal growth but suppresses development of conidia. Developmental activation necessary for asexual reproduction requires at least partial inactivation of FadA and this is executed by the product of another gene, flbA (YU et al. 1996). The FlbA protein has an RGS domain (for regulator of G protein signaling) that negatively regulates G-protein-mediated signaling pathways (DIETZEL and KURJAN 1987; DOHLMAN et al. 1995; DOHLMAN and THORNER 1997). Deletion mutations of flbA showed a phenotype similar to that of dominant activating mutations in fadA that caused failure of asexual sporulation accompanied by uncontrolled growth (LEE and ADAMS 1994; YU et al. 1996, 1999). Furthermore, both loss-of-function and dominant interfering mutations in fadA result in growth reduction and suppression of the developmental defects of flbA deletion mutant strains. On the other hand, deletion of the Gß-subunit encoded by sfaD, which acts not only as a regulator of the G-subunit but also as a signal transducer by itself, causes various phenotypes such as reduction of mycelial growth, hyperactive sporulation in submerged culture, and lack of cleistothecium formation (ROSéN et al. 1999).

The asexual spore conidium is critical for dispersal and constitutes a safe genomic storage device that can survive in adverse environments for filamentous fungi; thus spore germination is an essential developmental stage in the life cycle (D'ENFERT 1997; OSHEROV and MAY 2001). Whereas genetic and biochemical analysis of conidial development has been extensively studied, the signaling system for induction of conidial germination is not well understood (OSHEROV and MAY 2001). In A. nidulans, some components transmitting signals for germination have been characterized. It was reported that RasA, a small GTP-binding protein, is involved in conidial germination in A. nidulans (SOM and KOLAPARTHI 1994). Overexpression of a constitutively active form of RasA produced multinucleate swollen conidia that failed to form germ tubes, while overexpression of an inactivated form of RasA resulted in delayed germination. Also overexpression of an activated form of RasA induces germination without carbon source, indicating that RasA plays a role in carbon source sensing during conidial germination (OSHEROV and MAY 2000). Recently, the cAMP/PkaA pathway has been shown to be involved in conidial germination (FILLINGER et al. 2002). Deletion of cyaA, the adenylate cyclase gene, resulted in severe delays in germ tube formation, and inactivation of pkaA resulted in slower germ-tube-formation kinetics even though the degree of delay was less severe than that in the cyaA deletion mutant (FILLINGER et al. 2002). More recently in Penicillium marneffei, GasC, a homolog of the group III fungal G-protein, was shown to control conidial germination but was not involved in carbon source sensing. Deletion of GasC results in delay of conidial germination, and dominant activation of GasC causes accelerated germination (ZUBER et al. 2003).

The veA gene has been known to have dual functions in regulating developmental processes as a positive regulator of sexual and a negative one of asexual development (KIM et al. 2002). The veA1 mutant that was first isolated and characterized by KäFER (1965) reduced sexual development and increased conidium formation (CHAMPE et al. 1981). While the overexpression of veA reduced the formation of conidia, deletion of veA caused no cleistothecium formation even under conditions where sexual development preferentially occurs (KIM et al. 2002). These results suggest that VeA may have a key role in regulating both developmental processes.

To elucidate the signal transduction pathway regulating development of A. nidulans, we have isolated two genes, ganA and ganB, encoding G-protein homologs. Therefore there are at least three G proteins in A. nidulans. In this study, loss-of-function, overexpression, and constitutively active and constitutively inactive mutants of ganB were constructed and characterized. Phenotypes of these mutants suggested that GanB negatively regulates asexual development and plays a critical role in early events, including carbon source sensing during germination in A. nidulans. This is the first report that a G-protein is involved in carbon source sensing for germination in fungi.

Fungal strains, growth condition, and genetic manipulations:

A. nidulans RMS011 (STRINGER et al. 1991) from the Fungal Genetics Stock Center, Kansas City, and VER7 (HAN et al. 2001) were used as host strains for integration of manipulated genes. All A. nidulans strains used in this study (Table 1) were cultured on complex medium (CM) and minimal medium (MM). MM containing appropriate supplements was prepared as described (PONTECORVO et al. 1953; KäFER 1977) and CM was prepared by adding 1.5 g of casamino acids, 1.5 g of yeast extract, and 10 ml of vitamin solution per liter to MM. If necessary, 0.1% yeast extract or 0.1% casamino acids was added to MM. Transformation of Escherichia coli, plasmid DNA preparation, colony hybridization, Southern and Northern analysis, and other general molecular biological methods were as described in SAMBROOK and RUSSELL (2001). Transformation and genetic manipulations of A. nidulans were also as described elsewhere (PONTECORVO et al. 1953; YELTON et al. 1984; TIMBERLAKE 1990).

View this table: In this window In a new window   TABLE 1 A. nidulans strains used in this study

 

Isolation of the ganB gene:

The ganB gene was isolated using a fadA fragment (YU et al. 1996) as a heterologous probe. Southern analysis and colony hybridization showed that the chromosome VIII-specific genomic library (BRODY et al. 1991) contained the ganB gene. Two cosmid clones containing the ganB gene, W02C05 and W04H02, were isolated from this library. The cDNA of ganB, isolated by colony hybridization from an A. nidulans cDNA library in pBluescriptSK, were obtained from Jae Hyuk Yu (University of Wisconsin, Madison). Genomic DNAs and cDNAs of ganB were digested with various restriction enzymes, subcloned into pUC18 or pBluescriptSK, and sequenced.

Deletion of the ganB gene:

To isolate a ganB knockout mutant, a 1.4-kb fragment of the GanB open reading frame (ORF; Figure 1A) was removed by inverse PCR. Two primers were designed to amplify in an outward direction from the ganB ORF. Primer gbup1 was directed downstream of ganB and contained a SmaI site. Primer gblp2 was directed upstream of ganB and contained a ClaI site. The PCR product using these two primers contained the vector region plus insert, minus the 1.4-kb ganB ORF fragment. An 3.0-kb ClaI/SmaI fragment containing argB was ligated to the above PCR product that had been cut with ClaI/SmaI. The resulting plasmid, pdgBAR, was used as template in a PCR reaction amplifying from the outside the deleted ganB gene toward argB with another two PCR primers (gBpr7 and gBpr8). The PCR product with these two primers contained argB flanked on both sides by the remains of the ganB gene and was used to transform A. nidulans to obtain a knockout mutant. Knockout mutants of ganB were screened by PCR and Southern hybridization. We also confirmed by Northern analysis that the ganB gene was not expressed in these mutants.

View larger version (47K): In this window In a new window Download PPT slide   FIGURE 1.— The gene structure and transcription profile of ganB and the deduced amino acid sequence of GanB. (A) The gene structure and partial restriction map of ganB. The arrow indicates the direction of ganB transcription. Shaded boxes represent approximate exons of ganB ORF and interrupted regions indicate introns. Dashed line and dashed arrow indicate deleted region in the ganB mutant and the niiA promoter insertion in niiA(p)::ganB mutant, respectively. (B) The deduced amino acid sequence of GanB. Three regions within the GTPase domain, termed switch I, II, and III, are underlined. The amino acids R182 and Q208 (in the constitutively active mutations) and G207 (in the constitutively inactive mutation) that were substituted by site-directed mutagenesis are in boldface type and the myristoylation site is underlined. (C) The expression of the ganB gene during the A. nidulans life cycle. The mycelia of the wild-type strain cultured in CM broth for 14 hr were transferred to a MM plate (for inducing conidiation) containing 0.1% casamino acid (for inducing sexual development) and incubated for the time indicated above lanes. For sexual development induction, plates were tightly sealed with parafilm for 20 hr. The total RNA was isolated from mycelia of vegetative growth (lane 1, M), asexual development inducing (lanes 2–4) and sexual development inducing (lanes 5–10), and hybridized with a ganB-specific probe.

 

Isolation of ganB overexpression mutants:

The niiA promoter (PUNT et al. 1995) was isolated by PCR amplification with the primers nAup1 and nAlp3 and was then cloned into the AatII-XbaI sites of pGEM7ZF (Stratagene, La Jolla, CA). Plasmid pnAPGAR was constructed by inserting the argB gene into PstI-BamHI sites in the resulting plasmid. For ganB overexpression, the whole ganB ORF was isolated by digestion with BamHI and XbaI (Figure 1A) and cloned into BamHI-XbaI sites in pnAPGAR. The resulting plasmid (pOGB6) containing niiA(p)-ganB was introduced by homologous recombination into A. nidulans VER7 and RMS011. Mutants were screened by PCR and Southern blotting, and overexpression was confirmed by Northern analysis. Expression of ganB in these clones was induced by addition of 0.3% sodium nitrate and repressed by adding 0.2% ammonium tartrate as a nitrogen source (JOHNSTONE et al. 1990).

Construction of ganB constitutively active and inactive mutants:

To construct the constitutively active mutant of ganB, Gln208 was switched to Leu using site-directed PCR mutagenesis as described elsewhere (HO et al. 1989; KADOWAKI et al. 1989). For GanB^Q208L, we designed primer pair gBQ208Lup11 and gBQ208Llp12, containing sequences in which the CAG (Q) codon was changed into the CTG (L) codon and introducing an FspI site. PCR reaction I was performed with gBmbup7 and gBQ208Llp12, and PCR reaction II with gBQ208Lup11 and gBmblp8. A third PCR reaction was performed using PCR products I and II as a template to give a final product containing the Q208L mutation. After cloning this fragment into a vector containing the wild-type argB gene, the mutation site was confirmed by nucleotide sequencing the whole constitutively active mutant gene before transformation of A. nidulans. Screening and confirmation of mutant clones were performed by PCR, nucleotide sequencing, and Southern analysis after digestion of genomic DNA by FspI.

The procedure for constructing the constitutively inactive mutant was similar to that used for constitutively active mutants. GanB Gly207 was substituted with Arg. We introduced a NaeI (GCCGGC) site in primers gbG207Rup13 and gbG207Rlp14, which were used for switching codon GGC (G) to CGG (R). Again, mutant alleles were screened and confirmed by PCR, Southern hybridization, and nucleotide sequencing. The nucleotide sequences of all primers used in this study are listed in Table 2.

View this table: In this window In a new window   TABLE 2 Primers used in this study

 

Growth rate and conidiation:

We investigated growth rate by calculating radial colony extension and cell density of mycelia. After 2 µl of conidial suspension (1 x 10⁶conidia/ml) of wild-type and ganB mutant strains were point inoculated onto supplemented minimal medium plates, the suspension was incubated for 4 days, then mycelial blocks 0.5 cm in diameter were isolated from the colony margin, and 30 separated mycelial blocks were inoculated into 50 ml of liquid minimal medium in the 250-ml flask and incubated for 4 days, shaking at 80 rpm. Mycelial cultures were harvested, freeze dried, and weighed.

For radial extension measurements, conidial suspensions were point inoculated into the centers of minimal medium plates and incubated for 5 days before measuring colony diameter. Radial extension rate was determined as extension length per hour (in millimeters per hour) and the values were expressed as percentages of the wild type.

To count the number of conidia, conidia (1 x 10⁶) of wild type and ganB mutant strains were spread on minimal medium plates. After 5-day incubation, 0.8-cm agar blocks were vortexed in 1 ml of 0.08% Triton X-100 solution for 2 min and the resulting conidial suspension was counted with a hemacytometer. The number of conidiophores per square centimeter of medium plates was counted with a low-power microscope. The values for growth, conidia, and conidiophores are shown in Tables 3 and 4 as percentages of the wild type.

View this table: In this window In a new window   TABLE 3 Radial extension of colony and mycelial density in ganB mutants

 

View this table: In this window In a new window   TABLE 4 Conidiation of ganB mutants

 

Germination of conidia and ascospore:

Conidia of wild-type and mutant strains were inoculated into 20 ml of minimal medium in petri dishes containing autoclaved cover slips and incubated at 37°. We removed coverslips every 2 hr and observed swelling and germ tube emergence under the microscope. We also observed conidial germination of mutants in carbon-source-free medium.

To investigate germination of ascospores in ganB mutant alleles, wild-type and ganB mutants were incubated under sexual-development-inducing conditions for 10 days, when cleistothecia from each strain were isolated and suspended in 0.08% Triton X-100 solution. Cleistothecia and asci were broken with a mounted needle and vortexed. Ascospores released were inoculated into minimal medium with or without glucose and incubated as specified in the text.

The ganB gene encodes a G-protein homolog in A. nidulans:

The ganB gene (GenBank accession no. AF198116) has five introns in its ORF and one in the 5'-untranslated region (Figure 1A). The ganB gene located in chromosome VIII contains an open reading frame consisting of 356 amino acids that are perfectly matched to those of AN1016.2 in the A. nidulans genome database at the Whitehead Institute Center for Genome Research (WICGR; http://www.genome.wi.mit.edu/annotation/fungi/aspergillus; Figure 1B). GanB has conserved domains that are considered to be directly involved in interaction with the guanine nucleotide found in all G-proteins (SIMON et al. 1991; SKIBA et al. 1996; BOHM et al. 1997), as well as the myristoylation site in the N terminus (BUSS et al. 1987). The amino acid sequence of GanB shows 75–90% identity to the amino acids of GpaB (LIEBMANN et al. 2003) and GasC (ZUBER et al. 2003) of the human pathogenic fungi Aspergillus fumigatus and P. marneffei, respectively; to the amino acids of Cpg-2 (CHOI et al. 1995) and MagA (LIU and DEAN 1997) of the phytopathogenic fungi, Cryphonectria parasitica and Magnaporthe grisea, respectively; and to those of GNA-3 (KAYS et al. 2000) of N. crassa. When fungal G-proteins are classified according to amino acid sequence, these G-proteins belong to the group closely related to the mammalian G_s-subfamily that is involved in activation of adenylate cyclase (BöLKER 1998; ZUBER et al. 2002).

Northern blot analysis showed that the ganB transcript accumulated at higher levels during developmental stages than during vegetative growth, with greater levels during sexual development than during asexual development (Figure 1C).

Isolation of deletion, overexpression, constitutively active, and constitutively inactive mutants of the ganB gene:

To address the function of GanB in vivo, deletion, overexpression, constitutively active, and constitutively inactive mutants of ganB were isolated. To obtain deletion mutants, we constructed a plasmid in which the GanB ORF was deleted and argB substituted as a selectable marker (see MATERIALS AND METHODS). Because the presence of functional VeA affects the development pathway in A. nidulans, we isolated mutants from both strains of veA⁺ (VER7) and veA1 (RMS011). Eight ganB deletion mutants from VER7 and six from RMS011 were isolated and the deletion of ganB was checked by genomic Southern analysis. The fact that the transcripts of the ganB gene were not detected by Northern analysis of these mutants and sequencing the flanking region of inserted argB showed that the deletion was successful (data not shown).

We also isolated overexpression mutants of ganB using the niiA promoter that is induced in the presence of nitrate but repressed by ammonium (JOHNSTONE et al. 1990; see MATERIALS AND METHODS). Seven ganB overexpression mutants were isolated from VER7 and three from RMS011. Transcript levels for ganB were much higher in these mutants than in the parental strains VER7 and RMS011 (data not shown).

Gln208 in the switch II region and Arg182 in the switch I region of the GTPase domain of G protein (Figure 1) play important roles in the GTPase activity that hydrolyzes GTP bound to G protein to GDP (SIMON et al. 1991; SKIBA et al. 1996; BOHM et al. 1997). Mutations at either of these sites largely abolish the hydrolytic activity of intrinsic GTPase and thus stabilize the active, GTP-bound state (SIMON et al. 1991; COLEMAN et al. 1994). Many constitutively active mutants with substitutions of these amino acid residues have been isolated in a variety of fungal G proteins (WIESER et al. 1997; YANG and BORKOVICH 1999; YU et al. 1999; ZUBER et al. 2003). The constitutively active mutants (ganB^Q208L and ganB^R182L), in which the glutamine or arginine residue was substituted by leucine, were isolated by site-directed mutagenesis using PCR amplification (see MATERIALS AND METHODS). Ten ganB^Q208L constitutively active mutants from VER7 and 5 from RMS011 were isolated. In addition, 12 ganB^R182L mutants were isolated from VER7. Since ganB^R182L and ganB^Q208L mutants showed the same phenotypes, we here present the results from only the ganB^Q208L mutant as phenotypes of ganB constitutive activation.

In addition, we isolated constitutively inactive mutants by substituting arginine for Gly207. This glycine residue in the conserved DXXGQ motif of the switch II domain (Figure 1) has been proposed to act as a hinge involved in the conformational change that accompanies guanine nucleotide exchange (KURJAN 1992; BOHM et al. 1997). The Gly207Arg mutation prevents conformational changes to the GTP-bound G-subunit, resulting in inhibition of the release of Gß; the G protein thus stays in a constitutively inactive state (KURJAN 1992; YU et al. 1996; ZUBER et al. 2003). The difference between deletion and inactivating mutants of the G-protein is that the former releases the Gß-subunit but the latter does not. Because of this, signal transduction by Gß as well as by G is blocked in the inactivated mutant. We isolated six ganB^G207R inactivated mutants through site-directed mutagenesis of VER7 and RMS011 (see MATERIALS AND METHODS).

Constitutive activation of GanB causes reduction in mycelial growth:

To determine the effect of GanB on growth, we compared the growth rates of wild-type and ganB mutants by measuring cell mass or radial colony expansion. While cell mass and radial extension rates for deletion, constitutively inactive, and overexpression mutants of ganB were similar to those of the wild type, constitutive activation of GanB caused decreases in cell mass and radial extension to 69 and 74% of wild type, respectively (Figure 2 and Table 3). No differences in the effect of ganB mutations on growth were observed between veA⁺ and veA1 strains.

View larger version (153K): In this window In a new window Download PPT slide   FIGURE 2.— Phenotypes of ganB mutant strains. (A and C) Conidia of wild types (1 and 6, V7p16 and RMp16), ganB deletion mutants (2 and 7, V7dgB08 and RMdgB03), ganB constitutively inactive mutants (3 and 8, V7gBCI1521 and RMgBCI1633), ganB constitutively active mutants (4 and 9, V7gBQL705 and RMgBQL801), and ganB overexpression mutants (5 and 10, V7ogB105 and RMogB204) were point inoculated in the centers of minimal medium plates and incubated for 3 days at 37°. Note that the radial extension of the ganB constitutively active mutant is reduced. (B and D) The strains are the same as above and were grown on minimal medium plates for 3 days after spreading to observe asexual development. CP indicates conidiophore. Note that conidia are not observed in the picture of constitutively active mutants. Bar in B, 200 µm.

 

Absence or constitutive inactivation of GanB induces hyperactive sporulation and derepression of brlA in submerged culture:

To investigate the function of GanB in asexual development, we incubated ganB mutant strains in liquid minimal medium containing 0.1% yeast extract to observe conidiophore development in submerged culture where asexual development is normally suppressed in the wild type even after >24 hr incubation. It was reported that the addition of yeast extract or casamino acid to minimal medium increased growth and the number of conidiophores (ROSéN et al. 1999). The deletion and constitutively inactive mutants of ganB formed complete asexual conidiophores by 18 hr, whereas no development was seen in wild type, overexpression, or constitutively active mutants (Figure 3A). However, this submerged conidiation was observed only in a veA1 background, not in veA⁺ strains.

View larger version (77K): In this window In a new window Download PPT slide   FIGURE 3.— Deletion and constitutively inactive mutants of the ganB gene show hyperactive sporulation and derepress brlA expression in submerged culture. (A) Conidia of designated strains in a veA1 background were inoculated into 50 ml liquid minimal medium containing 0.1% yeast extract and incubated at 37°, shaking at 200 rpm. The photographs were taken of samples harvested at 18 hr. Arrows indicate complete conidiophores. (B) Total RNA was isolated from ganBQ208L (lanes 1 and 2), niiA(p)::ganB (lanes 3 and 4), wild type (lanes 5 and 6), ganB (lanes 7 and 8), and ganBG207R (lanes 9 and 10) grown for 12 hr (lanes 1, 3, 5, 7, and 9) and 18 hr (lane 2, 4, 6, 8, and 10) under the same conditions as above. The RNA blots were probed with a brlA-specific probe. Arrowhead in B indicates brlA transcript. Bar in A, 50 µm.

 
To determine whether the hyperactive sporulation phenotype observed in deletion and constitutively inactive GanB mutants was due to brlA expression, total RNA isolated from 18-hr submerged cultures was probed with a brlA-specific probe. The brlA transcript level was increased in deletion and constitutively inactive mutant strains, whereas no transcript was detected in the wild type (Figure 3B). The fact that the transcript of brlA was not detected in the submerged culture of any mutants in the veA⁺ background (data not shown) might explain why no submerged conidiation was observed in deletion or constitutively active mutants with veA⁺ background. These results suggest that activation of GanB may repress conidiophore formation in liquid culture and downregulate brlA transcript levels.

Activation or overexpression of GanB reduces conidiation on solid medium:

In surface cultures, formation of both conidiophores and conidia by constitutively active ganB mutants was dramatically decreased to <2.2% of the wild type in veA⁺ strains, with a slightly less marked reduction in conidophore formation in the veA1 background (Figure 2 and Table 4). Overexpression mutants also showed a reduction in conidiation but to a lesser extent than constitutively active mutants. Deletion or constitutive inactivation of GanB reduced conidiation to a certain extent on solid medium (Figure 2 and Table 4). This contrasts with the hyperactive sporulation phenotype of these mutants in submerged culture. Furthermore, conidiophore formation and the conidiation rate in the mutants having the veA⁺ allele is more aggravated, compared with the mutants having veA1 mutation. Especially, the relative conidiophore formation rate (6.7 ± 3.7%) of the ganB overexpression mutant to a wild type in the veA⁺ background is much lower than that (35.3 ± 1.5%) in the veA1 background (Table 4), suggesting that VeA may act synergistically with the GanB subunit to regulate asexual sporulation negatively. This is consistent with the suggestion of KIM et al. (2002) that veA may act as a negative regulator of asexual development.

GanB plays a crucial role in spore germination:

When conidia of deletion or constitutively inactive mutants of ganB were cultured in liquid medium, the number of mycelial balls was reduced relative to the conidium inoculum size (data not shown). This suggested that GanB might affect germination or conidial viability. To examine germination in various mutant strains, conidial swelling and germ tube emergence were observed under the microscope. Whereas conidial swelling and germ tube formation in deletion or constitutively inactive ganB mutants were delayed, those of constitutively active mutants were facilitated (Figure 4A). Wild-type conidia started to swell 2 hr after inoculation and only 8% of them had formed germ tubes by 4 hr. Meanwhile, 40% of conidia of constitutively active ganB mutants had germ tubes by 4 hr. By 6 hr, >90% of conidia of both wild type and the constitutively active mutant had formed the germ tubes. In contrast, very few conidia of deletion or constitutively inactive mutants had germ tubes by 4 hr, and only half of the conidia had produced germ tubes even after 10 hr. Deletion and constitutively inactive mutants also showed a highly asynchronous germination pattern (Figure 4B). These results imply that activation of GanB seems to play an important but dispensable role in early signaling for germination.

View larger version (41K): In this window In a new window Download PPT slide   FIGURE 4.— Conidial germination rate was delayed in ganB deletion and constitutively inactive mutants but was accelerated in a constitutively activate mutant. (A) Kinetics of germ tube outgrowth. Conidia of designated strains were inoculated into minimal medium and incubated at 37°. The number of conidia per microscope field showing germ tube was counted and presented as the percentage of total conidia in this field. The values are the mean of three independent experiments and ± represents standard error of the mean. (B) Deletion and constitutively inactive mutants of ganB showed asynchronous and delayed germination. Photographs were taken after 6 and 10 hr incubation at 37°. Bars in B, 50 µm.

 
We further examined the effects of ganB mutations on ascospore germination. Ascospores were inoculated into minimal medium containing glucose. As with conidia, the constitutive activation of GanB led to precocious ascospore germination while ganB deletion and constitutively inactive mutants germinated very poorly (data not shown). These results indicate that activated GanB is a positive signal transducer for initiation of germination in both kinds of spores.

Constitutive activation of GanB allows germination without carbon source:

When the constitutively active mutant form of RasA is overexpressed in A. nidulans, germinating conidia form multinucleate swollen spheres but fail to produce germ tubes (SOM and KOLAPARTHI 1994), and this occurs even in the absence of a carbon source (OSHEROV and MAY 2000). To examine whether ganB constitutively active mutants could germinate without carbon source, conidia of mutant strains were inoculated into carbon-free minimal medium. After 5 hr incubation most conidia of the constitutively active mutant formed germ tubes (Figure 5). In contrast, very few swollen cells or germ tubes were observed in wild type even after 24 hr.

View larger version (99K): In this window In a new window Download PPT slide   FIGURE 5.— Constitutive activation of GanB led to germination without carbon source. Conidia of wild type (WT) and the ganB constitutively active mutant (ganBQ208L) were inoculated into minimal medium without carbon source and incubated at 37°. Photographs were taken after 5 and 8 hr incubation. Bar, 40 µm.

 
In minimal medium without carbon source, ascospore germination showed a slightly different pattern from that of conidia. The germination rate of wild-type ascospores was comparatively higher (20–30%) than that of conidia under conditions without a carbon source. Whereas the germination rate of constitutively active mutant ascospores increased to 50% when compared to that of wild type, deletion or constitutively inactive mutants showed a germination defect as observed in conidia (data not shown).

Constitutive activation of GanB reduces hyphal growth:

G proteins, for example, FadA G-subunit and SfaD Gß-subunit of A. nidulans, are involved in regulating vegetative growth in fungi. To address whether GanB signaling regulates vegetative growth, we examined growth rate in ganB mutants constructed in this study. Of these, only constitutively active mutants showed any reduction in radial extension rate and cell mass when compared to other mutants and the wild type (Table 3 and Figure 2). The fluffy phenotype and hyphal autolysis observed in the constitutively active mutant of FadA (YU et al. 1996, 1999) was not seen in the equivalent mutant of GanB. The slight effect of ganB mutation on hyphal growth suggests that GanB may not be a key element regulating vegetative growth. Instead, GanB might act as a signal transducer in developmental processes that compete with hyphal growth. This hypothesis is supported by the phenotype of deletion or constitutively inactive mutants whose growth rate is not increased compared to the wild type.

Active GanB negatively regulates asexual sporulation:

When cultured on solid media, the number of conidiophores and conidia in ganB mutants was reduced (Table 4, Figure 2). The constitutively active mutant especially showed a severe decrease in conidiophore formation compared with other mutants, indicating that active GanB may be a negative regulator of conidiation. To confirm this hypothesis, we cultured ganB mutants in liquid medium where development, including conidiation, is normally suppressed. It was reported that starvation for carbon and/or nitrogen could induce submerged conidiation in A. nidulans (SKROMNE et al. 1995). Inappropriate submerged conidiation also occurs in mutants of genes involved in asexual development. For example, submerged conidiation was observed in a deletion mutant of sfaD (ROSéN et al. 1999), a dominant inactivating mutant of fadA (YU et al. 1996), and in an overexpression mutant of brlA (ADAMS et al. 1988). Deletion and constitutively inactive mutants of ganB also showed inappropriate submerged conidiation (Figure 3A). Since it seems likely that there are a Gß-subunit (AN0081.2), a G-subunit (AN2742.2), and three G-subunit homologs (AN0651.2, AN3090.2, and AN1016.2) in the A. nidulans genome database of WICGR (http://www-genome.wi.mit.edu/annotation/fungi/aspergillus), there may be three forms of heterotrimeric G proteins in this fungus. Whereas the constitutively inactive form of GanB is predicted to interfere with signaling by both the Gß-subunit and GanB itself, the deletion mutation of ganB is expected to interfere only with GanB signaling and not with Gß-signaling. We therefore suggest that the submerged conidiation observed in deletion and constitutively inactive ganB mutants is likely due to the lack of active GanB. This hypothesis is contrary to that proposed by the report of ROSéN et al. (1999) in which the absence of the SfaD Gß-subunit leads to submerged conidiation in liquid medium.

Recently, it has been reported that deletion of Gß causes a reduction in G-protein levels in several fungi, possibly by post-transcriptional regulation. In N. crassa, deletion of the Gß-gene, gnb-1, leads to downregulation of GNA-1, GNA-2, and GNA-3 G-proteins under a variety of medium conditions (YANG et al. 2002). Also, in C. parasitica, the level of the G-protein CPG-1 is greatly reduced in a deletion mutant of cpgb-1 that encodes Gß-protein (PARSLEY et al. 2003). These findings may give us an answer to the question of why a deletion mutant of sfaD causes submerged conidiation in liquid culture: the absence of Gß-signaling, caused by deletion of sfaD, may lead to a reduced GanB protein level, as in the situation described above for N. crassa and C. parasitica, and result in failure of active GanB to repress conidiation. However, we cannot rule out the possibility that the lack of GanB may reduce the Gß-protein level, resulting in submerged conidiation. Endpoint dilution immunoblot analysis showed that the protein level of the CPGB-1 Gß-subunit was slightly reduced in the absence of the CPG-1 G-subunit in C. parasitica and dramatically reduced in a double deficiency of CPG-1 and CPG-2 G-proteins (PARSLEY et al. 2003). These conditions lower the level of the G-subunit, resulting in submerged conidiation as in the sfaD deletion. We need more experiments to explain whether GanB alone, SfaD alone, or GanB and SfaD together negatively regulate asexual development.

Absence or inactivation of GanB derepresses the expression of brlA in a liquid culture:

The brlA gene is not transcribed under conditions where conidiation is not active (ADAMS et al. 1988) and if submerged conidiation observed in deletion or constitutively inactive ganB mutant strains is mediated through the BrlA pathway, brlA should be transcribed in liquid cultures of these mutants. As expected, brlA transcripts were detected only in deletion and constitutively inactive ganB mutants (Figure 3B). These results indicate that negative regulation of asexual sporulation by active GanB may be mediated through repression of brlA expression at the transcriptional level.

The veA gene encodes a putative transcription activator that may have dual functions in regulating developmental processes, acting as a positive regulator of sexual development and a negative regulator of asexual development (KIM et al. 2002). Submerged conidiation of ganB deletion or constitutively inactive mutants was not observed in strains carrying the veA⁺ allele but only in a veA1 genetic background, indicating that the veA gene is involved in negatively regulating submerged conidiation. Submerged conidiation has also been observed in a sfaD deletion mutant in veA1 but not in veA⁺ backgrounds (ROSéN et al. 1999). This suggests that the suppression of asexual development in submerged culture requires active GanB and VeA proteins.

GanB plays critical roles in the early events of spore germination:

In filamentous fungi, early signaling events in germination, including carbon source sensing, trehalose breakdown, swelling (isotropic growth), and germ tube emergence, are not well characterized because of the difficulty of dissecting germination at a molecular level (D'ENFERT 1997; OSHEROV and MAY 2001). In several filamentous fungi, germination may be controlled by multiple pathways, resulting in complication of genetic analysis. In addition to true early germination signaling events, essential metabolic and housekeeping activities occur at the beginning of the germination, making it difficult to differentiate between true signaling events and these activities (OSHEROV and MAY 2001). Nevertheless, in A. nidulans, some components of a conidial germination signal transduction system have been isolated and characterized. A RasA has been shown to have a function in carbon source sensing during conidial germination (SOM and KOLAPARTHI 1994; OSHEROV and MAY 2000). Recently, it was reported that cAMP/PkaA signaling plays an important role in conidial germination and it has also been shown that cAMP signaling and RasA act independently on conidial germination (FILLINGER et al. 2002).

Our results indicate that active GanB plays a critical role during early events of germination of both conidia and ascospores in A. nidulans. GasC, the GanB homolog of P. marneffei, plays a role in conidial germination (ZUBER et al. 2003). Unlike GanB, GasC does not seem to have a function of carbon sensing since conidia of the dominant activating mutant do not germinate in the absence of carbon source, suggesting that in this fungus there are separate mechanisms for carbon source sensing and for other early events of germination such as swelling and nuclear decondensation. Furthermore, the majority of ungerminated conidia of deletion or constitutively inactive ganB mutants cultured in minimal medium remained unswollen (data not shown), suggesting that GanB signaling is also involved in conidial swelling. Because the constitutively active ganB mutant showed successful swelling and germ tube emergence, and deletion or constitutively inactive mutants left many conidia unswollen, establishment of carbon source sensing is perhaps the earliest step in germination. Trehalose breakdown is defective in the ganB deletion mutant, suggesting that GanB signaling may play a role in the activation of adenylate cyclase (LAFON et al. 2003; C. D'ENFERT, personal communication). Taken together, these results lead to the conclusion that GanB signaling mediates early germination events in A. nidulans. Similar results were obtained with ascospores: ganB deletion and constitutively inactive mutants showed defects in swelling and germ tube formation (data not shown). Thus GanB signaling is involved in regulating germination of ascospores as well as of conidia.

Hypothetical model for the function of GanB:

The above results lead us to propose that GanB may function in signaling pathways of asexual development and germination (Figure 6). GanB negatively regulates asexual development by repressing brlA expression at least in submerged culture. In addition to GanB, the VeA protein is necessary to repress submerged conidiation possibly by repressing the brlA expression. GanB also positively controls early events, including carbon source sensing during germination. One of the candidates for GanB downstream targets in the signaling pathway regulating germination might be cAMP/PkaA, which is reported to have a function in conidial germination (FILLINGER et al. 2002).

View larger version (17K): In this window In a new window Download PPT slide   FIGURE 6.— Proposed model of the contribution of GanB in the signaling pathways of germination and conidiation. GanB negatively regulates asexual development by repressing brlA expression and also positively controls early events, including carbon source sensing during germination. The Gß-protein SfaD is known to be a positive regulator of growth and a negative one of submerged conidiation (ROSéN et al. 1999), which are depicted by double lines ending with an arrow and a bar, respectively. On the other hand, SfaD may function in maintaining the protein level of G (YANG et al. 2002; PARSLEY et al. 2003; also see DISCUSSION), which is represented by the dashed arrow.

 

We gratefully acknowledge Jae-Hyuk Yu (University of Wisconsin, Madison) for providing cDNAs, John Clutterbuck (Glasgow) for a critical reading of the manuscript, Tae-Ho Yang for drawing the hypothesis, and lab fellows for their encouragements and criticisms. This work was supported by grants from the Korea Science and Engineering Foundation [R01-1998-000-00053-0 (2001)] to K.-Y.J. and by a Korea Research Foundation grant (KRF 99-005-D00071) to K.Y.J.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AF198116.

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