INTRODUCTION

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G-protein-coupled receptors (GPCRs) are a family of seven transmembrane helix receptors that bind ligands such as neurotransmitters, pheromones, and odorants. GPCRs are associated with heterotrimeric G proteins, consisting of , , and  subunits (for a review, see reference 82). In the inactive state, the heterotrimer is docked at the receptor and GDP is bound to the G subunit. Binding of a ligand activates the receptor, leading to the exchange of GDP for GTP on G and the subsequent dissociation of G-GTP from the G moiety. G-GTP and the G dimer can activate downstream effectors, such as enzymes and ion channels, to produce a response to the signal (for a review, see reference 7). The response is terminated and the cycle is completed with hydrolysis of GTP by G. The GDP-bound G protein reassociates with G, and the heterotrimer is then able to bind to the receptor to await the next cycle of activation (for reviews, see references 10 and 20).

Neurospora crassa is a filamentous fungus that has a complex life cycle due to its ability to produce both asexual and sexual spores (for a review, see reference 71). Grown with adequate nitrogen, N. crassa remains in the asexual cycle and extends basal hyphae to form a complex, intertwined network called a mycelium. Asexual spores, or conidia, are produced by two pathways: macroconidiation and microconidiation. Both types of conidiation require an air-water interface and therefore are repressed in submerged culture. Macroconidiation is initiated by the production of specialized aerial hyphae above the surface of the agar. Conidiophores are formed at the tips of aerial hyphae and give rise to chains of multinucleate macroconidia. Microconidiation differs from macroconidiation in that uninucleate spores form inside the basal hyphae and burst through the hyphal wall upon maturation. In response to nitrogen-limiting conditions, N. crassa enters the sexual cycle by forming female reproductive structures called protoperithecia. Fertilization occurs when the nucleus from a conidium of the opposite mating type is taken up by a specialized hypha (trichogyne) emanating from the female structure. Subsequently, meiosis and sexual spore (ascospore) maturation occurs within the fertilized structure (perithecium).

Genes encoding two G subunits, gna-1 and gna-2, have been identified in N. crassa (79). The gna-1 strain has a decreased growth rate and reduced mycelial mass during vegetative growth, defects which are more pronounced under hyperosmotic conditions (32). In the sexual cycle, the gna-1 strain is female-sterile and forms aberrant perithecia after fertilization (5, 32). The gna-2 strain does not possess any phenotypes; however, gna-1 gna-2 strains are more impaired in gna-1 defects (5). Both gna-1 and gna-1 gna-2 strains have decreased intracellular steady-state cyclic AMP (cAMP) levels and adenylyl cyclase activity (33). Results of immunoinhibition experiments suggest that GNA-1 directly interacts with the adenylyl cyclase enzyme to regulate its activity (33).

Studies of G subunits in several saprophytic and pathogenic fungi have revealed roles for these proteins in the regulation of virulence, cAMP levels, and asexual and sexual development (for reviews, see references 8 and 38). Mutation of the G subunit gene mod-D in the saprophyte Podospora anserina results in impaired aerial hypha and mycelial development, defects which are repressed by exogenous cAMP (49). The P. anserina adenylyl cyclase gene, PaAC, has been cloned as a partial suppressor of the mod-D mutation (48). The mod-D strain is also defective in vegetative incompatibility through a cAMP-independent pathway (49). Deletion of any of the three Magnaporthe grisea G genes, magA, magB, or magC, reduces fertility. Perithecia fail to form after fertilization in the magB strain, and the magA and magC strains do not produce mature asci (44). Deletion of either magA or magC does not affect the pathogenicity of the organism; however, the magB strain displays reduced virulence. The magB strains have decreased appressorium formation, which is suppressed by exogenous cAMP. Two genes encoding G subunits, cpg-1 and cpg-2, have been cloned from Cryphonectria parasitica (24). The cpg-1 strains have reduced conidiation and 2.5-fold-higher cAMP levels, are female-sterile in heterozygous crosses, and are avirulent. The cpg-2 strain has only half the wild-type level of cAMP and is virulent (24). Four G genes have been cloned from Ustilago maydis, but only deletion of gpa3 produces an obvious phenotype. The gpa-3 mutants have elongated cells and are unable to respond to pheromone or to form the dikaryotic mycelium after fertilization (63). Both the cell morphology and mating defects are suppressed by exogenous cAMP (39). Deletion of gpa3 or of the catalytic subunit of the cAMP-dependent protein kinase gene, adr1, results in avirulence, suggesting that both participate in a cAMP signaling pathway (22, 63). In Ustilago hordei, loss of the G gene, fil1, impairs dimorphic switching between the sporulation and filamentous growth habits. This defect is overcome by supplementation with exogenous cAMP (43). Cryptococcus neoformans strains with the G gene, gpa1, deleted are mating deficient and avirulent. All mutant phenotypes in the gpa1 strain are suppressed by cAMP supplementation (2, 3).

Here we report identification of a new G gene from N. crassa, gna-3. The predicted protein sequence of GNA-3 is most similar to that of P. anserina MOD-D, M. grisea MAGA, and C. parasitica CPG-2. Phenotypes of gna-3 and cr-1 (an adenylyl cyclase mutant) strains were analyzed during the asexual and sexual cycles. We measured the levels of cAMP, adenylyl cyclase activity and protein, and transcripts for several genes in gna-3, cr-1, and wild-type strains. Our results revealed roles for gna-3 in the regulation of conidiation and the level of adenylyl cyclase protein in N. crassa.

	MATERIALS AND METHODS

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Strains, media and growth conditions. Wild-type strains 74-OR23-1A (abbreviated 74A; FGSC 987) and 74-OR23-1a (abbreviated 74a; FGSC 988) and the cr-1 B123A (FGSC 4008) mutant were acquired from the Fungal Genetics Stock Center (FGSC) at the University of Kansas Medical Center, Kansas City, Kans. Strains were cultured on either Vogel's minimal medium (VM) for vegetative growth or synthetic crossing medium (SCM) to induce the sexual cycle (19). Standing VM liquid cultures were supplemented with 1 mM cAMP (Sigma) or 2% (wt/vol) Bacto Peptone (Difco Laboratories, Detroit, Mich.) where indicated. For examination of hyperosmotic sensitivity, strains were grown on solid VM supplemented with 0.75 M NaCl, 0.75 M KCl, or 1.5 M sorbitol.

Isolation and sequencing of the gna-3 gene. Degenerate primers based on conserved regions of G proteins (79) were used in PCR with genomic DNA from a gna-1 gna-2 N. crassa strain (5). The resultant 250-bp gna-3 PCR product was subcloned into plasmid pBluescript II SK(+), yielding plasmid pGNA3. The pGNA3 insert was labeled (32) and used as a probe to screen the N. crassa -BARGEM7 genomic lambda library (59). A total of 4 positive plaques were obtained from 40,000 total screened. After conversion to double-stranded plasmids (59), the four clones were subjected to Southern analysis (32) using the pGNA3 insert as a probe. One of the clones (4-3b) was chosen for further analysis, since it contained the gna-3 amino acid coding region centered in an approximately 6.7-kb insert. A 4.35-kb region of the 4-3b insert extending from the extreme amino terminus to the third BamHI site (see Fig. 1; site B³) was sequenced (Core Sequencing Facility, Department of Microbiology and Molecular Genetics, University of TexasHouston Medical School). A full-length gna-3 cDNA was isolated by screening an N. crassa cDNA library made using RNA from SCM-grown cultures (55). The NsiI-XhoI fragment corresponding to the amino acid coding region was used as a probe. Sequencing was performed as described for the genomic clone. All sequence data were analyzed using Seqed (Applied Biosystems version 1.0.3) and the GCG program package (GCG Inc., Madison, Wis.).

gna-3 gene replacement mutation and complementation in trans. A gna-3 gene replacement plasmid (pSR20) was made that contains genomic DNA extending from the extreme 5' end of clone 4-3b to the KpnI site. Within this fragment, the region between the NsiI and XhoI sites was replaced by the EcoRV-BamHI fragment containing the bacterial hygromycin resistance gene (hph) from plasmid pCSN44 (72) (see Fig. 1). The pSR20 construct was linearized, and 2 µg was electroporated into 8-day-old conidia of 74A, as previously described (30, 80). Electroporation products were plated using regeneration agar (14) on sorbose medium containing 200 µg of hygromycin B per ml (32, 72). Genomic DNA from hygromycin-resistant strains was digested with EcoRI and subjected to Southern analysis (32) using a 1.8-kb KpnI-EcoRI fragment that lies outside of the genomic region in pSR20 as a probe. Heterokaryotic transformants containing nuclei with the gene replacement were crossed to wild-type strain 74a in order to isolate homokaryotic progeny. The homokaryotic gna-3 strains isolated were designated 2a1, 2a3, 2a17, 31c1, and 31c2 (all are MATA). A MATa gna-3 strain was obtained by crossing 31c2 to 74a and is designated 43c2.

Northern and RT-PCR analysis. The tissues used for RNA extraction were as follows. For reverse transcriptase PCR (RT-PCR) and cr-1 expression analysis, conidia were inoculated at 3.6 × 10⁶ conidia/ml in VM and cultured at 30°C in the dark for 16 h with shaking at 200 rpm. For analysis of con-10 and qa-2 expression, conidia were inoculated at 5.5 × 10⁵ conidia/ml and cultured at 30°C in the dark for 16 h with shaking at 200 rpm. For con-10 analysis, VM was the medium. For qa-2 expression studies, conidia were inoculated into VM and cultured for 16 h, at which time the mycelia were washed with sterile water and transferred to VM, VM plus 0.1% (vol/vol) quinic acid, or VM minus sucrose plus 0.1% (vol/vol) quinic acid (50). Total RNA was isolated as previously described (68). For Northern analysis, 30 µg of total RNA was electrophoresed and transferred (78) and the membrane was hybridized as described for Southern analysis (32). cr-1 was detected using a 1.25-kb SalI-PstI fragment from the region containing the catalytic domain (36). A 200-bp EcoRI-BamHI fragment (from pBW100) (64) was used as a probe for the con-10 gene, and a 237-bp SmaI-SphI fragment (pQA2-1088) (obtained from B. Tyler, University of California, Davis, Calif.) was used as a probe for the qa-2 gene (26, 50). A cosmid containing an rRNA gene (obtained from D. E. Ebbole, Texas A&M University) or the cox-5 gene (from pSRCOX-5) (68) was used as the loading control probe.

Western analysis. Submerged VM cultures were inoculated with 3.6 × 10⁶ conidia/ml and grown for 16 h at 30°C with shaking at 200 rpm. Plasma membranes were isolated as described previously (79), and protein was quantitated using the Bradford protein assay (Bio-Rad, Hercules, Calif.). Samples containing 30 µg of total protein were subjected to Western analysis (32). Primary antibodies against GNA-1, GNA-2, and GNB-1 were used at dilutions of 1:400, 1:200, and 1:5,000 (5, 32, 33), respectively. Detection was as described previously (32).

Phenotypic analysis. Apical extension rates were determined by measuring colony diameters at various times as previously described (32). For mating assays, strains were grown on SCM, with or without 2.5 mM cAMP, for 6 days at room temperature in constant light to induce the production of protoperithecia. Protoperithecia were fertilized using conidial suspensions from strains grown on VM. Cultures were incubated at room temperature for 7 days in constant light and then viewed and photographed using a SZH10 research stereoscope and a PM-20 exposure control unit (Olympus, Lake Success, N.Y.). Submerged cultures were inoculated using 5 × 10⁵ conidia/ml and cultured at 30°C for 16 h with shaking at 200 rpm in the dark (50). Samples were viewed using an Axioskop epifluorescence microscope (Zeiss, Thornwood, N.Y.), and images were digitally photographed using a DEI-750 color charge-coupled device camera (Optronics, Goleta, Calif.).

Measurement of intracellular steady-state cAMP levels and adenylyl cyclase activity. Samples from 16-h VM submerged cultures (grown as described above) or VM (in constant light for 3 days at room temperature) or SCM (in constant light for 6 days at room temperature) cellophane-overlaid plates were ground in liquid nitrogen and extracted in 6% trichloroacetic acid. Samples were centrifuged at 20,229 × g for 30 min at 4°C. cAMP in the supernatant was purified and quantitated as described previously (11, 33). Protein in the pellet was solubilized using 0.5% sodium dodecyl sulfate in 0.1 M NaOH and quantitated using the bicinchoninic acid protein assay reagent (Pierce).

Nucleotide sequence accession number. The accession number of the gna-3 genomic sequence is AF281862.

	RESULTS

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Identification of the N. crassa gna-3 gene. Previously, degenerate oligonucleotide primers designed to amplify conserved regions of mammalian G genes were used to isolate PCR products corresponding to gna-1 (approximately 200 bp) and gna-2 (approximately 250 bp) from N. crassa (79). A third N. crassa G gene, gna-3, was identified as an approximately 250-bp product present in PCR products containing genomic DNA from the gna-1 gna-2 strain (data not shown). The gna-2 and gna-3 products are larger than that of gna-1 due to the presence of an intron whose position is conserved in mammalian G_i and G_s family genes (Fig. 1) (79).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Gene structure of gna-3 from N. crassa. The shaded areas indicate the amino acid coding regions of gna-3 and hph, and arrows within these areas signify the direction of transcription. The translational initiation codon for gna-3 is indicated by +1. Intron positions (1, +125 to +183; 2, +533 to +619; 3, +749 to +809; 4, +939 to +994; 5, +1234 to +1293) are indicated by open triangles. Bars and arrows within the upstream region indicate pyrimidine-rich regions and consensus sites for binding by SRY family HMG box proteins, respectively. The dashed line at the NsiI and XhoI sites illustrate the region of gna-3 that was replaced by hph. Restriction sites: K, KpnI; B, BamHI; C, ClaI; E, EcoRV; X, XhoI, and N, NsiI.

View larger version (127K): [in this window] [in a new window]   FIG. 2.   Amino acid sequence alignment of G proteins homologous to GNA-3 from filamentous fungi and yeasts. Alignment was performed using the GeneDoc program. Dark and light shading indicate identical and similar amino acids, respectively. Abbreviations and accession numbers: C. parasitica (Cp) CPG2, L32177; M. grisea (Mg) MAGA, AF011340; P. anserina (Pa) MODD, AF038122; N. crassa (Nc) GNA3, AF281862; U. maydis (Um) GPA3, U85777; U. hordei (Uh) Fil1, U76672; C. neoformans (Cn) GPA1, U09372; S. cerevisiae (Sc) GPA2, S50478; and S. pombe (Sp) GPA2, 2832746.

Deletion of gna-3 by targeted gene replacement. A gna-3 mutant was isolated after electroporation of a wild-type strain with a construct in which the gna-3 amino acid coding region was replaced by the bacterial hygromycin B resistance gene (hph⁺; Fig. 1). Genomic DNA from hygromycin-resistant transformants was subjected to Southern analysis following digestion with EcoRI (Fig. 3A). In this way, heterokaryotic strains containing both wild-type (corresponding to a 7.0-kb fragment) and gna-3 (4.1-kb fragment) nuclei were identified. Homokaryotic gna-3 strains were obtained by crossing heterokaryotic primary transformants to the wild type, with selection for growth of progeny on hygromycin B. Replacement of the gene in all nuclei was confirmed using Southern analysis (Fig. 3A).

View larger version (24K): [in this window] [in a new window]   FIG. 3.   Southern and Western analyses. (A) Southern analysis. DNA from the wild type (WT, strain 74A) and from the indicated gna-3 homokaryotic strains was digested with EcoRI and subjected to Southern analysis using a KpnI-EcoRI fragment of gna-3 as a probe. The sizes of hybridizing fragments are indicated by arrows on the left. (B) Quantitative RT-PCR. Reaction mixtures contained total RNA from the wild-type strain, 74A (WT) or from gna-3 (strain 31c2), gna-3 + gna-3+, or cr-1 strains or a plasmid containing the gna-3 cDNA (positive control). Southern analysis was performed using the NsiI-XhoI region of the gna-3 amino acid and coding region as a probe. The position of the 1.1-kp hybridizing RT-PCR product is shown by the arrow. (C) Western analysis. Samples containing 30 µg of plasma membrane protein were subjected to Western analysis using antiserum directed against GNA-1, GNA-2, or GNB-1, as indicated on the left. Strains are the same as in panel B.

gna-3 strains have reduced fertility during homozygous crosses. The G proteins GNA-1 and GNA-2 contribute to female fertility in N. crassa, and their homologues have also been shown to regulate sexual fertility in filamentous fungi (5, 8, 32, 38). Therefore, the gna-3 strains were examined for defects during the sexual cycle. When cultured under nitrogen-limiting conditions (SCM), gna-3 strains produced protoperithecia in amounts similar to those produced by wild-type and gna-3 + gna-3⁺ strains and were fertile when used as males or females in heterozygous crosses (data not shown). Perithecial formation was observed 2 days after fertilization and ascospores were produced within 11 days in both wild-type and gna-3 strains (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 4.   Sexual-cycle phenotypes. Protoperithecia from strains 74A and gna-3 + gna-3+ (MATA) were fertilized with a 74a conidial suspension, while those from strain gna-3 (strain 31c2, MATA) were fertilized with a gna-3 (strain 43c2, MATa) conidial suspension. Perithecia were photographed 7 days after fertilization. Arrows indicate perithecia embedded in the agar.

Hyperosmotic sensitivity. Deletion of gna-1 results in decreased growth rate and mycelial mass during vegetative growth; these properties are more prominent on hyperosmotic media (32). The gna-1 gna-2 strain is more impaired in this trait, indicating overlapping functions (5). To determine whether GNA-3 contributes to osmosensitivity, the apical extension rate of gna-3 strains was compared to that of the wild type on minimal medium (VM) plates with and without the hyperosmotic agents 0.75 M NaCl, 0.75 M KCl, or 1.5 M sorbitol (data not shown). Relative to the wild type, the growth rate of gna-3 strains was 78 to 83% on VM, 69 to 74% on VM-NaCl, and 69 to 73% on VM-KCl. The gna-3 strain was more sensitive to 1.5 M sorbitol, with growth rates of 57 to 61% of that of the wild type. Therefore, deletion of gna-3 has only a slight effect on apical extension rates on VM hyperosmotic media, with the greatest effect observed during growth on sorbitol. This is in contrast to gna-1 strains, which display much greater and almost equal sensitivity to NaCl, KCl, and sorbitol (32).

Asexual growth and development of gna-3 mutants. During vegetative growth on solid medium, aerial hyphae grow up from the basal hyphae and produce conidiophores, which terminally differentiate at the tips to yield macroconidia. In contrast to wild-type strains, gna-3 mutants produce short aerial hyphae and conidiate prematurely, yielding a dense conidiation pattern on VM plates and in VM standing liquid cultures. Ectopic insertion of gna-3 complements the defects in aerial hypha formation and conidiation (data not shown).

View larger version (72K): [in this window] [in a new window]   FIG. 5.   Analysis of standing liquid and solid medium cultures. (A) Standing liquid cultures. Tubes contain liquid VM with no additions or with added 2 mM cAMP or 2% (wt/vol) peptone, as indicated. The gna-3 strain is 31c2. Cultures were incubated at 30°C for 3 days in the dark followed by 4 days at room temperature in the light. (B) Growth on solid medium. Plate cultures on VM with or without 2.5 mM cAMP were photographed after incubation at room temperature for 4 days in the light. The gna-3 strain is the same as in panel A.

gna-3 strains conidiate in submerged culture. Given an air-water interface, wild-type N. crassa strains produce conidiophores; conversely, conidiation is normally repressed in liquid submerged cultures. However, carbon or nitrogen starvation induces wild-type strains to conidiate in submerged culture (18, 28, 50, 61, 77). To explore another possible role for gna-3 during conidiation, we analyzed submerged-culture growth morphology. We found that 16-h submerged cultures of wild-type strains produced long, slender hyphae that intertwined to form a mycelium with no conidiophores (Fig. 6A). In contrast, both the gna-3 and cr-1 strains formed conidiophores in submerged culture, with a greater amount being formed in gna-3 strains. Supplementation with cAMP did not affect the submerged-culture conidiation phenotype of gna-3 and cr-1 strains (data not shown). The wild-type strain grown in submerged cultures containing peptone produced swollen hyphae but was otherwise unaffected (Fig. 5A). The gna-3 strain containing the ectopic copy of gna-3 did not conidiate in submerged culture and responded to cAMP and peptone supplementation in a manner similar to the wild-type strain (Fig. 5A and data not shown). Although swelling of the hyphae was observed, peptone addition repressed conidiation in both the gna-3 and cr-1 strains.

View larger version (57K): [in this window] [in a new window]   FIG. 6.   Submerged-culture conidiation. (A) Morphology. Cultures were grown in liquid VM with or without peptone at 30°C for 16 h in the dark with shaking. Arrows indicate conidiophores. The gna-3 strain is 31c2. (B) Analysis of con-10 expression. Samples containing 20 µg of total RNA were subjected to Northern analysis, using the con-10 gene as a probe. The cox-5 gene probe was used as a loading control. The gna-3 strain is the same as in panel A. (C) Analysis of qa-2 expression. Samples containing 25 µg of total RNA isolated from strains cultured under the indicated conditions were analyzed on Northern blots by hybridization with the qa-2 gene. The blot was hybridized with an rRNA gene to check the relative amount of RNA in each lane. The gna-3 strain is the same as in panel A. WT, wild type.

gna-3 strains have decreased steady-state intracellular cAMP levels and adenylyl cyclase activity. Previous analysis of cr-1 mutants has shown that exogenous cAMP suppresses phenotypic defects and that these strains contain no detectable adenylyl cyclase activity (66, 67, 73, 74). Based on the similar morphological phenotypes and response to cAMP observed in cr-1 and gna-3 strains, intracellular steady-state cAMP levels of wild-type, gna-3, and cr-1 strains were measured under various growth conditions.

gna-3 strains have normal cr-1 transcript levels but reduced CR-1 protein levels. The mechanism by which GNA-3 regulates the expression of adenylyl cyclase was explored by measuring the amount of cr-1 transcript and protein present in gna-3 strains. Although different amounts of RNA were present, Northern analysis indicated that the 7.0-kb cr-1 mRNA could be detected in all strains (Fig. 7A). Production of a cr-1 transcript has previously been observed for the cr-1 B123A allele (36, 54). Significantly, total RNA and cr-1 mRNA levels were similar for the gna-3 and wild-type strains. Observance of wild-type levels of the cr-1 transcript in gna-3 strains suggests that GNA-3 does not regulate the transcription of adenylyl cyclase.

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Analysis of cr-1 transcript and protein levels. (A) Northern analysis. Samples containing 20 µg of total RNA were analyzed for the presence of the cr-1 transcript (top) using a gene fragment from the catalytic domain of cr-1 as a probe. Hybridization with cox-5 (bottom) was performed to determine the relative amount of RNA in each lane. The gna-3 strain is 31c2. (B) Western analysis. Samples containing 30 µg of total membrane protein from 16-h germlings were subjected to Western analysis using the CR-1 antibody (top). The position of the major immunoreactive species is shown on the right. The gna-3 strain is the same as in panel A. A duplicate gel was Coomassie stained to check protein loading (bottom). The sizes of molecular mass standards are indicated on the right. WT, wild type.

	DISCUSSION

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We have investigated the importance of heterotrimeric G-protein-mediated signal transduction systems in the response of N. crassa to environmental cues. The G protein GNA-1 has been shown to participate in mating and stress response pathways and to overlap functionally with a second G, GNA-2 (5, 32). Analysis of adenylyl cyclase activity supports the hypothesis that GNA-1 is a direct positive regulator of this enzyme in N. crassa (33). Here we have reported the identification and functional analysis of a third N. crassa G subunit gene, gna-3. One wild-type allele of gna-3 is necessary for successful completion of the sexual cycle. GNA-3 negatively regulates conidiation through a cAMP-dependent pathway in standing liquid cultures and on solid medium. Relative to the wild type, both cr-1 and gna-3 strains have greatly elevated levels of a glucose-repressible gene when starved for carbon. Examination of a role for GNA-3 in adenylyl cyclase regulation demonstrates that GNA-3 influences cAMP levels by posttranscriptionally controlling the amount of adenylyl cyclase protein.

Deletion of gna-1 results in the production of very small aberrant perithecia, which rarely produce ascospores (32). Loss of gna-2 does not independently affect mating; however, the gna-1 gna-2 strain displays a more severe defect in female fertility with complete absence of perithecia and ascospores (5). The defect in female fertility caused by the deletion of gna-1 suggests that GNA-1 may participate in the N. crassa pheromone response pathway by coupling to a pheromone receptor. Results of studies of gna-2 support a minor role for this G in pheromone sensing. In contrast, gna-3 strain perithecia are smaller than wild-type perithecia, lack beaks, are often embedded in the agar, and produce few viable ascospores during homozygous crosses. The differences between gna-1, gna-2, and gna-3 mutants suggest that various aspects of mating, such as fertilization, perithecial formation, and production of sexual spores, are independently regulated.

Differential regulation of the sexual cycle by G subunits is also observed in M. grisea and C. parasitica. Regulation of mating is similar in M. grisea and N. crassa in that the gna-1 homologue, magB, and the gna-3 homologue, magA, regulate similar aspects of mating (44). The gna-2 homologue, magC, appears to have overlapping functions with magA rather than magB, in that both the magA and magC mutants display the same defects. Homologues of gna-1 (cpg-1) and gna-3 (cpg-2) have been identified in C. parasitica, and only the deletion of cpg-1 impairs sexual development (24). Phenotypic similarities resulting from mutation of these G proteins suggest that a common mode of regulation exists for monitoring the sexual cycle in these related filamentous fungi.

The loss of gna-3 leads to decreased aerial hypha height and premature conidiation in standing liquid cultures as well as to inappropriate conidiation in submerged cultures. These results suggest a role for GNA-3 as a crucial negative regulator of conidiation in response to specific environmental and/or growth cues. One activating signal for conidiation is blue light (57); however, gna-3 strains grown in standing liquid cultures or on solid medium prematurely initiate conidiation even in the dark (data not shown). A similar phenotype is observed in the adenylyl cyclase mutant, cr-1 (73). Exogenous cAMP suppresses the premature and dense conidiation defect of both gna-3 and cr-1 strains in standing liquid and plate cultures. These findings suggest that under these growth conditions, conidiation is mediated by a cAMP-dependent mechanism.

Submerged-culture conidiation is induced by carbon starvation in N. crassa (18, 28, 50, 61, 77). The submerged conidiation phenotype of cr-1 and gna-3 strains is similar to that observed in rco-3 mutants (50). Despite the high sequence similarity of RCO-3 to a S. cerevisiae glucose transporter, RCO-3 has been proposed to act as a glucose sensor because both high- and low-affinity transport are affected in rco-3 strains (50). The abundant submerged conidiation of the rco-3 strains, like that observed for the cr-1 and gna-3 strains, is suppressed by peptone. However, unlike gna-3 strains, rco-3 does not display conidiation defects on solid medium and is sorbose resistant (reference 50 and data not shown). The qa-2 gene is expressed in rco-3 to levels comparable to those in carbon-starved wild-type cultures, independent of the carbon status. Expression of qa-2 is significantly higher in cr-1 and gna-3 strains than in the wild type in submerged culture under carbon-limiting conditions. The higher levels of qa-2 expression is negatively influenced by cAMP levels during carbon starvation. This is the first time that cAMP levels have been shown to affect the transcription of qa-2. Future work investigating the relationships between rco-3, gna-3, and cr-1 will facilitate the elucidation of carbon sensing in N. crassa.

In the filamentous fungus A. nidulans, induction of conidiophore development under normal growth conditions requires exposure to air and can be induced by transferring cultures to media lacking either carbon or nitrogen (4, 70). Deletion or dominant interfering mutations of the G subunit gene, fadA, or a loss-of-function mutation in a regulator of G protein signaling gene, flbA, results in inappropriate submerged-culture conidiation (30, 84). Both fadA mutations suppress the flbA mutation, indicating that FlbA negatively regulates the activity of FadA (84). When cultures of the flbA mutant are transferred to media lacking a carbon or nitrogen source, conidiation does not occur, suggesting that FlbA is necessary in the response to carbon and nitrogen starvation (41). Although FadA is more similar to N. crassa GNA-1 than to GNA-3, the observed phenotypes of fadA mutants suggest that G-protein-mediated regulation of conidiation is a common theme in filamentous fungi.

Nutrient limitation induces morphogenesis in many species and is mediated by G proteins in other fungi, including S. cerevisiae. Diploid S. cerevisiae cells switch from budding to pseudohyphal growth during nitrogen starvation (for reviews, see references 9, 62, and 76). Deletion of either GPA2 or RAS2 reduces pseudohyphal development; loss of both genes results in a severe growth defect with an additive effect on filamentation (40, 45). Studies have shown that the high-affinity ammonium permease, Mep2p, is required for pseudohyphal growth; however, alternative nitrogen sources are still capable of being transported into the cell by other permeases (46). A GPCR, Gpr1p, has been identified by yeast two-hybrid analysis using Gpa2p as bait, indicating that these two proteins interact (83, 85). The gpr1/gpr1 diploid is defective in pseudohyphal growth. Transcription of GPR1 is induced in response to nitrogen starvation, and Gpr1p is subsequently required for sensing the presence of fermentable sugars, leading to increased cAMP levels (37, 47). Both the mep2/mep2 and the gpr1/gpr1 switch defects are suppressed in the presence of dominant-active GPA2 or RAS2 mutations or exogenous cAMP (46, 47, 85). Studies with the gpa2/gpa2 strain have shown that Gpa2p is necessary for glucose-induced accumulation of cAMP (17). Increased levels of cAMP activate the cAMP-dependent protein kinase (PKA) catalytic subunit, Tpk2p, which is a positive regulator of filamentous growth (60, 65).

The different effects of exogenous cAMP during sexual and vegetative development in gna-3 strains indicate that GNA-3 participates in both cAMP-dependent and -independent pathways. Likewise, P. anserina MOD-D functions in cAMP-dependent and -independent pathways (48, 49). Similar dual functions have also been observed with Gpa2p in S. cerevisiae. As mentioned above, Gpa2p is required for glucose-induced increases in cAMP. Recently, an interaction between GTP-bound Gpa2p and the meiosis/sporulation protein kinase, Ime2p, has been shown to inhibit Ime2p kinase activity (21).

Loss of cpg-2 or magA does not affect pathogenesis, but deletion of gpa3, gpa1, and fil1 renders the respective organism avirulent. The intracellular cAMP levels are altered or the mutant phenotype is corrected by exogenous cAMP in many cases, predicting that these G proteins control cAMP levels, presumably by directly regulating adenylyl cyclase activity (8, 15, 16, 38, 63). However, the activity and/or protein levels of adenylyl cyclase have not been investigated in these various organisms. Previous studies from our laboratory show that GNA-1 is a direct, positive regulator of adenylyl cyclase activity in N. crassa. Deletion of GNA-1 results in an 85% decrease in Mg²⁺-ATP-dependent adenylyl cyclase activity, and addition of a GNA-1 antibody specifically inhibits this activity in wild-type cells (33). The work presented here demonstrates that GNA-3 is not a direct stimulator of adenylyl cyclase but, instead, regulates the amount of the enzyme present in the organism. Therefore, two different G proteins, GNA-1 and GNA-3, coordinate the amount of positive stimulation and the level of the enzyme, respectively, leading to dual regulation of adenylyl cyclase in N. crassa.

This study demonstrates that GNA-3 posttranscriptionally regulates the level of adenylyl cyclase to mediate vegetative development in N. crassa. The high level of similarity in amino acid sequence and function suggests that G proteins homologous to GNA-3 may share a comparable mode of adenylyl cyclase regulation in filamentous fungi. This system of regulation could be compared to what is known about adenylyl cyclase in diverse fungal species. In S. cerevisiae, adenylyl cyclase activation requires Gpa2p and Ras2p and binding of the cytosolic cyclase-associated protein (CAP) to the C-terminal region of the adenylyl cyclase protein, Cyr1p (23, 81). cyr1 mutants arrest at the G₁ phase of the cell cycle in the absence of exogenous cAMP. In contrast, adenylyl cyclase is dispensable in the fission yeast S. pombe (51-53). CAP is required for adenylyl cyclase activity, and mutation of gpa2 results in decreased intracellular cAMP levels and a failure to produce cAMP in response to glucose (31, 35). Genetic studies with S. pombe have shown that Gpa2 acts upstream of adenylyl cyclase; however, direct regulation of adenylyl cyclase by Gpa2 has not been demonstrated (56, 76). The amino acid sequences encoded by the M. grisea and U. maydis adenylyl cyclase genes, MAC1 and uac1, respectively, are most similar to CR-1 from N. crassa (1, 27). Deletion of MAC1 has a dramatic effect on conidiation, growth rate, sexual development, and appressorium formation, and MAC1 can complement the N. crassa cr-1 mutation (1). The uac1 mutant is defective in dimorphic switching and maintains constitutive filamentous growth (27). In contrast to S. cerevisiae, adenylyl cyclase is not essential for cell viability in N. crassa, M. grisea, U. maydis, and S. pombe, suggesting a divergence in the physiological role of cAMP between budding yeast and other fungi (1, 27, 51-53, 66, 73). Based on the current model for regulation of adenylyl cyclase in all of these fungal systems, we predict that Ras and possibly CAP will be important in the regulation of adenylyl cyclase in filamentous fungi.

Our data support PKA as a central component in numerous developmental pathways in N. crassa (Fig. 8). The PKA regulatory subunit, mcb, was previously identified and shown to regulate cell polarity during vegetative hyphal growth (13). The catalytic subunit of PKA has recently been isolated, and a deletion mutant has been constructed (M. Plamann, personal communication). Deletion of the catalytic subunit results in a strain with phenotypes identical to those of the cr-1 strain. The common phenotypes observed for this mutant and the cr-1 and gna-3 strains suggest that PKA is responsible for positively regulating the extension of basal hyphae and the formation of aerial hyphae while negatively regulating conidiation. Our work further indicates that a cAMP-dependent pathway facilitates glucose-repressible gene expression under carbon-limiting conditions. RCO-3 has been previously demonstrated to negatively mediate submerged-culture conidiation and glucose-regulated gene expression (50). At present, possible links between RCO-3 and GNA-3 pathways have not been determined.

View larger version (35K): [in this window] [in a new window]   FIG. 8.   Model for action of GNA-3 in N. crassa. Control of adenylyl cyclase protein levels by GNA-3 and GTP-dependent stimulation by GNA-1 results in regulation of intracellular cAMP levels. Binding of cAMP to PKA results in dissociation of the MCB (regulatory) and PKA-C (catalytic) subunits. Activated PKA-C positively regulates aerial hypha formation and apical extension of basal hyphae and negatively regulates aerial and submerged conidiation and glucose-regulated gene expression. GNA-3 also regulates ascospore production through a cAMP-independent pathway. RCO-3 has previously been shown to negatively influence submerged conidiation and glucose-regulated gene expression (50). A G protein, GNB-1, has been identified in N. crassa, and its function is currently being investigated (Q. Yang, S. I. Poole, and K. A. Borkovich, unpublished data). Gray arrows indicate steps that may involve intermediate components. Question marks indicate unknown ligands for GPCRs coupled to GNA-1 and GNA-3.

Much work has been done to elucidate roles for G subunits in virulence in pathogenic fungi. Most of these studies have specifically addressed the correlation of reduced pathogenicity with the loss of a G protein. Deletion of G subunits leads to morphological and mating defects that compromise the ability of the fungus to invade and infect its host. Frequently, pathogenicity is restored by supplementing the mutant with exogenous cAMP, suggesting regulation of adenylyl cyclase activity as a role for G subunits related to GNA-3 (8, 38). Understanding the events that occur downstream of these heterotrimeric G proteins could lead to the construction of an appropriate pharmacological agent to disrupt the ability of pathogenic fungi to cause infection. The high degree of sequence and functional conservation between GNA-3 homologues suggests that concepts elucidated during studies in N. crassa will be widely applicable to understanding virulence in filamentous pathogens.