Article

• The EMBO Journal (1999) 18, 5592 - 5600
• doi:10.1093/emboj/18.20.5592

The Aspergillus nidulans sfaD gene encodes a G protein subunit that is required for normal growth and repression of sporulation

Stefan Rosén^1,2, Jae-Hyuk Yu^1,3 and Thomas H. Adams^1,3

1. Department of Biology, Texas A&M University, College Station, TX 77843, USA
2. Present address: Department of Microbial Ecology, Lund University, Ecology Building, Sölvegatan 37, S-223 62 Lund, Sweden
3. Present address: Cereon Genomics, LLC, 45 Sidney Street, Cambridge, MA 02139, USA

Correspondence to:

Thomas H. Adams, E-mail: Tom.H.Adams@cereon.com

Received 19 May 1999; Accepted 31 August 1999; Revised 27 August 1999

• Keywords:

  • Aspergillus nidulans,
  • conidiophore,
  • growth regulation,
  • heterotrimeric G protein,
  • RGS domain

Introduction

The balance between growth, sporulation and mycotoxin (sterigmatocystin) production in the filamentous fungus Aspergillus nidulans is controlled by the genes flbA and fadA. FlbA is an RGS (regulator of G protein signaling) domain protein that is required to suppress growth signaling by FadA, the subunit for a heterotrimeric G protein. This FlbA-dependent inhibition of FadA signaling is required for sporulation and sterigmatocystin (ST) production to occur (Yu et al., 1996; Hicks et al., 1997; Adams et al., 1998). Both genetic and biochemical experiments using several different RGS domain proteins have demonstrated that the major role for this conserved domain is to regulate G protein signaling negatively by facilitating the intrinsic GTPase activity of the G subunit (Dietzel and Kurjan, 1987; Berman et al., 1996; Koelle and Horvitz, 1996; Yu et al., 1996). The three-dimensional structure of a complex between the RGS protein RGS4 and G_i1 (Tesmer et al., 1997), as well as other recent studies (Hepler et al., 1997; Yan et al., 1997), suggest that some RGS proteins also can function as effector antagonists that compete for effector binding to G.

The role of flbA in controlling growth and asexual sporulation was clarified by analyzing epistatic interactions between flbA and fadA gain- and loss-of-function mutations (Figure 1). Dominant-activating mutations in fadA that are predicted to eliminate the intrinsic GTPase activity of this G subunit (fadA^G42R, fadA^R178C and fadA^Q204L) cause the same phenotype as loss-of-function mutations in flbA: a block in both asexual and sexual sporulation accompanied by uncontrolled growth that leads to proliferation of undifferentiated hyphal masses that autolyse as the colonies mature (Lee and Adams, 1994a; Yu et al., 1996, 1999). In contrast, both loss-of-function and dominant interfering mutations in fadA (fadA^G203R) resulted in reduced growth and suppressed the developmental defects of flbA deletion mutant strains. These results are consistent with the hypothesis that the normal role of FlbA in controlling sporulation is to inactivate FadA-dependent growth signaling (Yu et al., 1996).

Figure 1.

FlbA and FadA have a major role in controlling A.nidulans growth and development. We have previously proposed that two antagonistic signaling pathways regulate growth and asexual development in A.nidulans. Activated FadA G protein (GTP bound) affects downstream effector molecules to increase growth and repress sporulation. Either constitutive activation of FadA (G42R, Q204L, R178C, R178L) or loss-of-function FlbA result in a fluffy non-sporulating and autolytic phenotype. FlbA is an RGS domain protein that functions as a GTPase-activating protein (GAP) to inactivate FadA (GDP bound). This inactivation of FadA then allows asexual development to occur. However, because forced expression of flbA resulted in inappropriate spore formation even in a fadA deletion mutant, FlbA must have additional function in activating asexual sporulation. Developmental signals that are necessary for both activation of FlbA and asexual development require the wild-type FluG activities. The relevant mutant phenotypes are described.

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While many aspects of the requirement for FlbA in sporulation could be explained through its role in inactivating FadA, we also found that overexpression of flbA could cause inappropriate activation of asexual development and ST production both in wild-type and fadA deletion strains. This result raised the possibility that FlbA could have other targets in addition to FadA that must be affected to allow sporulation and ST production (Yu et al., 1996; Hicks et al., 1997). In this respect it is interesting to note that the fadA^G203R dominant-interfering mutation not only suppressed loss-of-function mutations in flbA but also caused hyperactive asexual sporulation and resulted in production of asexual sporulation structures, called conidiophores, even under environmental conditions that blocked wild-type sporulation. Because deletion of fadA did not cause hyperactive sporulation and the fadA^G203R mutation is predicted to inactivate signaling by preventing dissociation of active, GTP-bound, G protein from the G subunits, one possible second target for FlbA could be the G dimer (Yu et al., 1996). However, it is also possible that the secondary role of FlbA is to inactivate G proteins other than FadA.

In order to understand better the complex role of FlbA in controlling growth, development and ST production, we isolated extragenic suppressors of loss-of-function mutations in flbA (Yu et al., 1999). Two genes identified by these mutations, sfaC and sfaD, were of particular interest, because mutations suppressed loss of FlbA function and caused constitutive sporulation in submerged culture similar to that observed with fadA^G203R dominant-interfering mutants. Here we show that sfaD is predicted to encode the subunit of a heterotrimeric G protein. Characterization of sfaD mutations and their interactions with flbA and fadA support the hypothesis that both SfaD and FadA are positive growth regulators that negatively affect asexual and sexual development in A.nidulans. FlbA is required for controlling the activities of both FadA and SfaD.

The A.nidulans sfaD gene encodes a G subunit for a heterotrimeric G protein

Five different genes (designated sfaA–sfaE) were defined previously as extragenic suppressors of loss-of-function mutations in flbA (Yu et al., 1999). We showed that one of these suppressors (sfaB) resulted from a novel dominant-negative mutation in fadA (fadA^R205H) and proposed that one target for other suppressor mutations could be genes encoding the or subunits that associate with FadA. Because a previously defined dominant-negative fadA mutation (fadA^G203R) caused the abnormal phenotype of asexual sporulation during growth in submerged culture, we were particularly interested in further characterizing two flbA suppressor mutations, sfaC and sfaD, which caused a similar submerged sporulation phenotype. Both sfaC and sfaD mutations were dominant, which led us to attempt to clone the mutant genes by constructing cosmid libraries with genomic DNA from either sfaC or sfaD mutants and using these to transform an flbA98; argB2 mutant strain (fluffy autolytic), followed by visual screening to identify transformants that conidiated like wild type. However, after examining >3000 transformants each with sfaC67 and sfaD82 libraries, no suppressed flbA transformants were identified.

In order to test the hypothesis that sfaC, sfaD, or one of the other suppressor loci might encode the G subunit for a heterotrimeric G protein, we identified a gene encoding a G subunit by using PCR with degenerate primers designed around conserved sequences in known G proteins (Materials and methods). This approach resulted in the isolation of a gene with an open reading frame (ORF) predicted to encode a 352 amino acid polypeptide (Figure 2A) that shares 60% identity with mammalian G proteins. This A.nidulans ORF was most similar (82% identity) to Cpgb-1 from Cryphonectria parasitica (Kasahara and Nuss, 1997), the only other G subunit so far reported from a filamentous fungus, but shared much less similarity (40% identity) with the Saccharomyces cerevisiae G homologue Ste4p. As with other G subunits (Wall et al., 1995; Sondek et al., 1996), the A.nidulans ORF (Figure 2B and C) included seven highly conserved 40 amino acids repeats terminated by a Trp–Asp dipeptide (so-called 'WD40 repeats').

Figure 2.

The sfaD gene structure. (A) A restriction map of a 3.2 kb PstI fragment containing the sfaD gene is shown. The direction of sfaD transcription was deduced by analysis of cDNA clones and is indicated by the arrow positioned above the map. Approximate intron positions are indicated by discontinuities in the arrow. The location of the sfaD coding region is represented by the black box. The XhoI site just upstream of initiation ATG and downstream of stop codon was introduced by site-directed mutagenesis and used for constructing the sfaD deletion plasmid, pSRBD1. Restriction sites are abbreviated: A, ApaI; Bg, BglII; K, KpnI; P, PstI; R, EcoRI; S, SmaI; V, EcoRV; X, XhoI. (B) The deduced amino acid sequence of SfaD. The figure shows the alignment of the seven WD repeats in SfaD with amino acids predicted to be members of a structural triad of amino acids and the trademark tryptophan, indicated by plus signs (Sondek et al., 1996). Mutations in the different alleles of sfaD are underlined and introduced amino acids after conversion are in bold. An asterisk indicates an introduced stop codon; fs, frame shift. (C) A schematic representation of the different mutant alleles of the SfaD protein compared with wild-type SfaD. The seven WD repeats are boxed. Corresponding amino acid changes and nucleotide changes are shown where A of the ATG is indicated as 1. The DDBJ/EMBL/GenBank accession No. for the sfaD nucleotide and peptide sequences is AF056182.

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Because we initiated these experiments to learn whether any of the mutations we identified based on their ability to suppress loss-of-function flbA mutations corresponded to a gene encoding the G subunit, it was of interest to learn whether a G deletion mutation could suppress a flbA-null mutation. To test this, we first constructed an A.nidulans strain (TSRB1.16) lacking the G-encoding gene (G; see Materials and methods). TSRB1.16 was then meiotically crossed with a flbA mutant to generate flbA;G double mutant strains. As shown in Figure 3, the flbA;G mutant sporulated almost as wild type, indicating that the G deletion suppressed the sporulation defect of flbA mutants.

Figure 3.

Phenotypes of sfaD mutant strains. (A) The wild-type (FGSC26), (B) flbA (RJH046), (C) sfaD (RSRB1.15) and (D) sfaD;flbA (RSRF1.34) strains were point inoculated onto minimal medium (Käfer, 1977) and allowed to grow for 3 days at 37°C. (E and F) Wild-type (FGSC 26) and sfaD (RSRB1.15) strains after 10 days of growth. At this time point the sfaD deletion strain (F) showed a dramatically increased number of Hülle cells formed compared with wild type (E). These Hülle cells, which are structures proposed to function in supporting fruiting body formation in A.nidulans, formed white fluffy aggregates on the surface of the colony. Lower panels represent a 30–fold magnification of the colonies in upper panels. Arrowheads in the lower panel in (B) indicate areas where a flbA strain has started to autolyse.

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The question of whether a previously defined flbA suppressor mutation could represent an allele of the G-encoding gene was addressed by sexually crossing a flbA;G mutant strain (RSRF1.31) with flbA^- strains having sfaA^s, sfaC^s, sfaD^s or sfaE^s suppressor mutant alleles. Approximately 25% of the progeny from crosses with RSRF1.31 and flbA^-;sfaC^s or flbA^-;sfaE^s had the flbA^- phenotype (fluffy autolytic non-sporulating progeny) indicating that these sfa mutations were not linked to the G-encoding gene. We were unable to cross RSRF1.31 with the flbA^-;sfaA^s; however, no fluffy progeny were recovered from crosses between RSRF1.31 and various sfaD^s mutant strains, indicating that these sfaD suppressor mutations could represent alleles of G.

To address further the question of equivalence for sfaD and the G-encoding gene, we next constructed genomic DNA libraries from each of five different sfaD mutant strains (sfaD82, sfaD123, sfaD125, sfaD126 and sfaD127) and isolated the G-encoding genes by hybridization (Materials and methods). Because we had previously shown that each of these sfaD^s alleles was dominant to wild type under normal growth conditions at 37°C, each G clone from the different sfaD^s mutants was used to transform a flbA^- mutant strain. More than half of the transformants using the G clones from the sfaD123 and sfaD127 mutant strains sporulated like wild type, supporting the hypothesis that sfaD encodes the putative heterotrimeric G protein subunit. In contrast, all of the transformants using G clones from sfaD82, sfaD125 and sfaD126 had a flbA^- phenotype, indicating that either the mutant alleles were not dominant in a merodiploid or sfaD does not encode the putative G protein. Finally, we sequenced the G-encoding gene from each sfaD^s mutant strains and found that each strain had different mutations in the G-encoding gene, indicating that sfaD indeed encodes the putative G protein (Figure 2).

As shown in Figure 2, the sfaD123 and sfaD127 mutant alleles, which each behaved as dominant mutations in the transformation assay described above, result from missense mutations. For sfaD123, the highly conserved aspartate at position 258 in WD-repeat number five was converted to an asparagine, while for sfaD127, a cysteine at position 176 in WD-repeat three was changed to tyrosine. In contrast, the sfaD82, sfaD125 and sfaD126 mutant alleles that behaved as recessive mutations in the transformation assay all represent nonsense or frameshift mutations. The sfaD82 and sfaD125 mutations introduced stop codons at amino acid 138 (TCA to TAA) and 109 (TGG to TAG), respectively. The sfaD126 had an insertion of a cytosine converting arginine 312 to proline and introducing a frameshift in the ORF that leads to early termination at amino acid 333. This allele was distinct in that its ability to suppress flbA^- mutants was cold sensitive; it could suppress the flbA^- phenotype at 37°C but not at 25°C.

Because previous data indicated that mutations in all of the different sfaD alleles described here were dominant in diploids that were heterozygous for sfaD and homozygous for flbA (e.g. sfaD82;flbA/sfaD⁺;flbA) when grown at 37°C (Yu et al., 1999), yet sfaD82, sfaD125 and sfaD126 behaved as recessive mutations in transformation experiments, it was of interest to investigate the dominance relationships of sfaD mutations in diploid strains. Diploids were generated from heterokaryons formed between RSRF1.31 (sfaD;flbA) and RJH046 (sfaD⁺;flbA) and the resulting diploid strain (sfaD;flbA/sfaD⁺;flbA; DSRGF1.1) was found to sporulate, although it was partially fluffy. This indicates that deletion of sfaD is partially dominant, perhaps due to altered ratios of G protein , and subunits.

SfaD and FadA both mediate growth signaling

As shown in Table I, the radial extension rate for sfaD deletion mutant strains grown on solid medium was similar to that observed for wild type, although the hyphae were less dense (Figure 3). In submerged cultures, the sfaD mutant grew significantly slower than wild type with biomass accumulating to only 20% of wild-type levels (Table I). Biomass accumulation for submerged cultures of fadA strains was also reduced significantly, to 63% of wild-type levels. The biomass of a sfaD;fadA double mutant strain was nearly the same as a sfaD mutant, 20% of wild-type levels. While the conidiation defect of flbA mutants was suppressed by either sfaD or fadA, flbA suppressed the growth defect observed in fadA but not sfaD mutants. These results support the hypothesis that both FadA and SfaD are required for normal growth but that these activities are at least somewhat distinct in their requirements for FlbA.

Table 1: Phenotypic characteristics of fadA, flbA and sfaD mutant strains

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The fadA gene was originally identified by a dominant-activating mutation that is predicted to inactivate its intrinsic GTPase activity and lock FadA in a constitutive signaling GTP-bound state. Such mutations result in a fluffy phenotype much like that observed in flbA mutants and characterized by increased hyphal growth with a complete absence of sexual and asexual sporulation. To test whether this phenotype was dependent on the presence of an intact G protein subunit, we transformed a sfaD mutant with fadA^R178C or fadA^Q204L dominant-activating alleles. Several strains containing the wild-type fadA gene and one or more copies of either of these dominant-activating alleles were recovered. In addition, one strain was isolated where the wild-type fadA gene had been replaced with the fadA^R178C allele. All strains that had acquired a dominant-activating fadA allele were fluffy, non-sporulating and autolytic, as was expected if loss of sfaD did not influence the mutant phenotype. The inability to isolate asexual spores from these mutants prevented analysis of biomass accumulation in submerged culture.

SfaD-mediated growth signaling negatively affects sporulation

Wild-type A.nidulans strains do not sporulate in submerged culture under normal growth conditions (Adams et al., 1998). However, our previous results showed that sfaD82 and sfaD125 mutant strains were able to sporulate in submerged culture, and Figure 4 shows that sfaD mutant strains also elaborated nearly complete conidiophores during growth in submerged culture by between 19 and 22 h post-inoculation. This phenotype was not dependent on either fadA or flbA, because sfaD;fadA and sfaD;flbA double mutants also sporulated with identical timing under submerged growth conditions (Figure 4; Table I). As shown previously, sfaD123, sfaD126 and sfaD127 mutant strains did not produce conidiophores in submerged cultures.

Figure 4.

A complete loss-of-SfaD function eliminates the need of air and FlbA activity for conidiophore development. Conidia (1 10⁶ conidia/ml) from (A) wild type (FGSC26), (B) flbA (RJH046), (C) sfaD (RSRB1.15) and (D) sfaD;flbA (RSRF1.31) were inoculated into 100 ml of supplemented liquid minimal media (Käfer, 1977) containing 1 g yeast extract per liter in 250 ml flasks and incubated at 37°C shaking at 300 r.p.m. Mycelia from each culture were observed microscopically every hour starting at 12 h of growth (final observation at 26 h) to assess development. Micrographs were taken at 17 h when sfaD deletion (C) and sfaD; flbA (D) strains formed conidiophores (arrowheads). Wild-type (A) and flbA (B) strains never produced conidiophores under these conditions. All strains tested had a mutation in the veA gene (veA1). The scale bar shown in (A) is 20 m.

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While sfaD mutants always sporulated in submerged culture, we found that the composition of the growth medium had interesting effects on the timing and extent of conidiophore development by sfaD mutant strains. During growth in standard minimal nitrate medium (Käfer, 1977), the sfaD deletion strains occasionally developed complete conidiophores between 19 and 22 h post-inoculation (Table II). Addition of 10 or 100 mg/l of either yeast extract or casein hydrolysate (Difco Laboratories) to the minimal medium resulted in increased growth and caused a 30- to 300–fold increase in the number of conidiophores produced (Table II). Because yeast extract and casein hydrolysate have in common an abundance of amino acids that could serve as a complex nitrogen source, we examined the potential for better defined nitrogen sources to enhance submerged growth and development. As shown in Table II, we found that inclusion of 6 mM ammonium tartrate enhanced both growth and sporulation. In contrast, both glutamine and asparagine provided enhanced growth but did not stimulate conidiophore formation. For all media tested, the pH of the growth medium varied between 6.2 and 6.7 at 19 h post-inoculation. To ensure that differences in development did not result from differences in pH, we altered the initial pH of the medium between pH 5.7 and 7.2, but did not observe any significant effect on the number of conidiophores formed.

Table 2: Media composition affects submerged conidiation and growth of sfaD deletion strains

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SfaD and FadA are required for normal sexual development

Aspergillus nidulans reproduces sexually by forming ascospores in fruiting bodies called cleistothecia, which typically are surrounded by specialized globular cells, called Hülle cells, that are proposed to function in supporting cleistothecial development (Yager, 1992; Navarro-Bordonaba and Adams, 1994). As shown in Table III, we found that mutations in fadA and sfaD had major effects on sexual development. Deletion of sfaD resulted in an 22–fold increase in Hülle cell formation relative to wild type (Figure 3F; Table III). However, no cleistothecia were ever produced. Similarly, the fadA^G203R dominant-negative mutation had an 9–fold increase in Hülle cell formation and cleistotheciation was completely blocked. In contrast, deletion of fadA did not result in increased Hülle cell formation compared with a wild-type strain, although these mutants did fail to form cleistothecia. sfaD;fadA double mutants had an intermediate phenotype producing a 13–fold increase in Hülle cells and blocking cleistothecia. Finally, strains with dominant-activating fadA mutant alleles did not form Hülle cells or cleistothecia, regardless of sfaD genotypes, and the colonies were completely autolysed after prolonged incubation.

Table 3: Mutations in sfaD and fadA affect sexual development

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Although little is known about what controls sexual reproduction in A.nidulans, one gene that has been shown to influence cleistothecium formation is veA. Commonly used laboratory strains of A.nidulans, including those described here, have the veA1 mutation, which has the effect of removing a light requirement that is normally present for asexual development and leading to enhanced asexual sporulation while reducing cleistothecium production (Mooney and Yager, 1990). To examine the effect of the wild-type veA gene on sexual and asexual sporulation by sfaD mutants, sfaD;veA⁺ strains were generated and compared with sfaD;veA1 mutants (see Materials and methods). We generally found that asexual sporulation was reduced in sfaD;veA⁺ strains and that mutation in the veA gene (veA1) is required for hyperactive sporulation in sfaD deletion strains. No conidiophores were observed in sfaD;veA⁺ strains in submerged cultures, regardless of illuminating conditions. Furthermore, the biomass increased in sfaD;veA⁺ strains compared with sfaD;veA1 strains from biomass 19 1.4% (mean SE) to 61 2.2% of wild-type (sfaD⁺) biomass (n = 12). The biomass for the sfaD⁺;veA1 (FGSC26) and the sfaD⁺;veA⁺ (FGSC4) wild-type strains did not differ significantly from each other. The radial growth rates for sfaD;veA⁺ strains were similar (0.44 0.08 mm/h using minimal medium, n = 4) to sfaD;veA1 strains (see Table I).

We showed previously that fadA encodes the subunit of a heterotrimeric G protein in A.nidulans, which upon activation stimulates proliferation and antagonizes multiple processes including conidiation and secondary metabolism (Yu et al., 1996; Hicks et al., 1997). FadA signaling is inactivated by FlbA, an RGS domain protein that is proposed to function by stimulating the GTPase activity of FadA. The data presented in this paper demonstrate that the sfaD gene, which was identified as a suppressor of flbA loss-of-function mutations, probably encodes the subunit for a heterotrimeric G protein. As detailed below, we propose that the G heterodimer containing SfaD is an active participant along with the FadA encoded G protein in signaling downstream effectors that determine critical decisions in the A.nidulans lifecycle and differentiate multiple processes including growth, asexual and sexual development, and secondary metabolite production (Figure 5).

Figure 5.

Coordinate control of A.nidulans growth and asexual development. We propose that both SfaDG and FadA function in response to unknown growth signals to stimulate growth in liquid submerged culture and for aerial growth (plate). FlbA is required for controlling the activities of both FadA and SfaDG. Our study supports that both SfaDG and the veA gene product mediate signaling that negatively regulate asexual development. In air exposed cultures, red light-dependent derepression of the activity of the veA gene product is required for optimum asexual development. In submerged cultures asexual development requires inactivation of both the veA and sfaD gene products.

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While loss-of-function mutations in fadA were previously shown to cause significant reductions in hyphal growth, we found that this effect was enhanced by deletion of sfaD. sfaD mutants were as strongly restricted in growth as sfaD;fadA double mutants. Although these results are consistent with SfaD having a direct role along with FadA in activating effectors that signal hyphal growth, there are at least three complicating factors clouding this interpretation. First, FadA dominant-activating mutations are not at all affected by sfaD mutations (Table I), indicating that FadA can function independently of SfaD in causing uncontrolled proliferative growth that leads to autolysis and cell death. This finding both confirms our earlier conclusion that FadA actively controls proliferation (Yu et al., 1996) and shows that whatever the role of SfaD in proliferative growth, it is not essential.

The second complicating factor in interpreting the effects of sfaD mutations on growth results from the knowledge that the A.nidulans genome, like that of other filamentous fungi, has at least two additional genes encoding heterotrimeric G protein subunits (our unpublished data). In contrast, sfaD appears to be the only gene encoding a G protein, although there are numerous genes encoding members of the WD40-repeat protein family. Thus, SfaD probably interacts with other G proteins, and it is possible that some effects of deleting sfaD result from altering activity of distinct signaling pathways. No mutations have been described to date for these additional A.nidulans G-encoding genes but in other filamentous fungi, mutations in homologous genes have little or no phenotypic effect (e.g. Gao and Nuss, 1996; Liu and Dean, 1997; Regenfelder et al., 1997). In any case, the fact that loss-of-function mutations in either fadA or sfaD caused suppression of flbA loss-of-function mutations supports the conclusion that FadA and SfaD function as part of the same heterotrimeric G protein.

The final complication in interpreting the role of SfaD in growth comes from the observation that deletion of sfaD (but not fadA) resulted in hyperactive sporulation defined by sporulation in submerged culture, a growth condition that normally blocks development. Thus, one role of effectors stimulated by the G heterodimer could be suppression of the pathway leading to asexual sporulation during submerged growth. This interpretation is further supported by earlier findings that dominant-negative mutations in fadA, which are proposed to prevent dissociation of the G and G subunits, also caused hyperactive sporulation (Yu et al., 1996). Previous studies have shown that misactivation of genes with specific roles in activating sporulation (e.g. brlA or flbD) caused a severe reduction in fungal growth (Adams and Timberlake, 1990; Wieser and Adams, 1995). One possible interpretation of these results is that SfaD functions predominantly in activating a signaling pathway that prevents asexual sporulation and the effect of deleting sfaD on growth is entirely indirect. This interpretation might explain the different growth responses observed for fadA;flbA and sfaD;flbA double mutant strains (Table I). Because deletion of flbA did not suppress the hypersporulation phenotype of sfaD mutants it was not able to suppress the growth defect completely. Finally, it is important to recognize that loss of sfaD is not sufficient to allow asexual sporulation to occur. Loss-of-function mutations in sfaD were unable to suppress mutations in the development-specific regulator FluG (data not shown; Lee and Adams, 1994b, 1996).

While asexual and sexual development are clearly distinct processes in the life cycle of A.nidulans, some earlier studies have indicated that these two important events must have certain features in common. For instance, many mutations that were originally identified as causing defects in conidiophore development also block sexual development (Yager, 1992; Navarro-Bordonaba and Adams, 1994). We have shown here that mutations affecting both sfaD and fadA cause profound effects on sexual development along with their effects on growth and asexual sporulation. Loss-of-function mutations in sfaD and dominant-inactivating mutations in fadA both resulted in abundant production of Hülle cells, which are normally produced during formation of sexual fruiting bodies and are thought to have a supporting function in fruiting body formation (Yager, 1992; Navarro-Bordonaba and Adams, 1994). We interpret this result to mean that sfaD signaling negatively impacts Hülle cell development in much the same way as we propose it affects asexual sporulation. In contrast, we found that sexual fruiting body formation was blocked by loss-of-function mutations in sfaD or fadA or dominant-activating mutations in fadA. Thus, controlled signaling by FadA and SfaD is in some way required for further sexual development.

The relationship between sfaD and sexual or asexual development is further complicated by our finding that the ability of sfaD mutations to cause hyperactive sporulation was suppressed by the wild-type allele of a gene called velvet (veA⁺). Most laboratory strains of A.nidulans have the veA1 mutation, which causes increased asexual sporulation at the expense of sexual sporulation. VeA has been proposed to function as a negative regulator of conidiophore development that prevents asexual sporulation in dark-grown colonies of A.nidulans. VeA is inactivated by red light, allowing sporulation to occur in light–grown colonies (Mooney and Yager, 1990). The mechanism by which asexual sporulation is blocked by VeA is not clear, but our results suggest a model in which VeA and SfaD block development through independent but partially redundant mechanisms that monitor environmental conditions. If VeA is absent, SfaD is sufficient to block hyperactive or submerged sporulation although light control has been removed. When SfaD is absent, VeA maintains light control of conidiation by air-exposed colonies and provides an alternative block to hyperactive sporulation during submerged growth. When both SfaD and VeA are absent, conidiation occurs inappropriately both in the dark and in submerged culture.

Cpgb-1 from the chestnut blight fungus Cryphonectria parasitica is the only G subunit that has previously been described for a filamentous fungus and is the closest known relative to SfaD, having 82% identical amino acids. As with A.nidulans, C.parasitica apparently has a single gene encoding a G protein subunit, but at least two G protein subunits (cpg-1 and cpg-2). Cpg-1 is closely related to FadA and like fadA, null mutants in cpg-1 have reduced growth as well as having reduced virulence in C.parasitica pathogenesis of chestnut trees (Choi et al., 1995; Gao and Nuss, 1996; Yu et al., 1996). Interestingly, deletion of cpgb-1 from C.parasitica caused the opposite phenotype of A.nidulans sfaD deletion: increased growth rate and reduced asexual sporulation (Kasahara and Nuss, 1997). This difference in regulatory outcomes for what is apparently a highly conserved fungal signaling pathway may well reflect innate differences in the lifestyles of A.nidulans and C.parasitica. The asexual sporulation pathway is normally activated quite early in growth of an A.nidulans colony while active growth is still abundant, but in C.parasitica asexual sporulation takes place after growth is exhausted. As this pathway is understood in more detail and in more fungi it will be interesting to discover how subtle changes in responses lead to vast differences in timing and regulation of critical life cycle events in different fungi.

Aspergillus strains, growth conditions and genetics

The A.nidulans strains used in this study are described in Table IV. Standard A.nidulans transformation and genetic techniques were used (Pontecorvo et al., 1953; Yelton et al., 1984). When appropriate, genotypes of strains generated for this study were confirmed by genomic DNA Southern blot analyses. The sfaD deletion strains TSRB1.16 and TJYGV2 were generated by transforming PW1 and VER7, respectively, with pSRBD1. The VER7 strain was a gift from Dong Min Han, Wonkwang University, South Korea. RSRB1.4, RSRB1.15 and RSRB1.19 were derived from a sexual cross between FGSC237 and TSRB1.16. sfaD;flbA (RSRF1.31), sfaD;fluG (RSRG1.1) and sfaD;fadA^G203R (RSRR1.21) strains were generated by sexual crosses of TSRB1.16 with RJH057 (flbA), TTA127.4 (fluG) and RJY115.33 (fadA^G203R), respectively. RSRFA.1 (sfaD;fadA) was generated from a sexual cross of RSRB1.4 with RJY 918.12 (fadA). The sfaD;fadA^Q204L strain (RSRFG4) was generated by transforming RSRB1.4 with a plasmid carrying the fadA^Q204L allele (pJY.26SH) and was shown to have at least two copies of the fadA^Q204L allele as well as wild-type fadA⁺. The sfad;fadA^R178C strains (RSRGD2 and RSRGD6) were generated by transforming RSRB1.19 with pJY.27SH and were shown to have a replacement of wild-type fadA (RSRGD6) or a single integration of the fadA^R178C allele at the trpC locus (RSRGD2). To test whether a loss-of-function mutation in G could be dominant, diploids were generated between RSRF1.31 (sfaD;flbA) and RJH046 (sfaD⁺;flbA).

Table 4: Aspergillus nidulans strains

View full table

All strains were grown in appropriately supplemented minimal or complete medium (Käfer, 1977) unless otherwise indicated. Radial growth rates were determined as described previously (Wieser et al., 1994). For biomass analyses and developmental timing in submerged cultures, liquid cultures were inoculated at a density of 1 10⁶ spores/ml (100 ml medium/250 ml Erlenmeyer flasks). The flasks were incubated on a New Brunswick rotary shaker (300 r.p.m.) at 37°C and mycelia from each culture were observed microscopically every hour starting at 12 h to assess development. To determine biomass, cultures were harvested by filtering through Miracloth, freeze-dried and weighed.

Nucleic acid manipulations and other methods

Cloning of the gene encoding the G subunit (sfaD) was as follows. The degenerate primers pGB5 (5'–ATHTAYGCNATGCAYTGG–3') and pGB3 (5'–RAARTCRTCRTANCCNGC–3'), with H = A + C + T, R = A + G, Y = C + T and N = A + T + G + C, were used for PCR amplification of A.nidulans DNA from wild-type strain FGSC26. Amplification was achieved in 35 cycles of 0.5 min at 94°C, 1 min at 43°C, and 1.5 min at 72°C. The PCR product was precipitated, run on an agarose gel and a 0.75 kb fragment was excised. The labeled fragment was used to screen a partially ordered genomic cosmid library and three cosmid clones known to be derived from chromosome eight (W08C10, W17E07 and W30E09) were recovered (Brody et al., 1991). A 3.2 kb PstI fragment from one of these cosmids (W17E07) was cloned into pBluescript SK^- for sequencing (pSRB10). Four incomplete G cDNA clones were isolated by screening the UNI-ZAP library made from vegetatively grown wild-type tissue (May et al., 1987). The 5' end of G cDNA was PCR amplified using 5'–CGACGCGCTCGTTCCGC–3' and 5'–GGACTTTGTTCGTATGG–3' as primers with gt10 (Osmani et al., 1988) cDNA library as a template. A 0.4 kb amplified fragment was isolated, cloned into pPK1 and sequenced.

The plasmid used for deletion of G, pSRBD1, was constructed from pSRB10. The XhoI and SalI sites (found in the polylinker region) in pSRB10 were eliminated by digestion with XhoI and SalI and religation resulting in plasmid pSRB11. Using this plasmid the entire ORF (25 bases upstream and 91 bases beyond G start and stop codon, respectively) of G was deleted using site-directed mutagenesis with the synthetic oligonucleotide 5'-CAATGCGGACATCCGGCTCTCGAGCGGCAAT- TGGAGTGGTG-3', as described previously (Kunkel, 1985), introducing a XhoI site 25 bases 5' of the G start codon. The resulting plasmid, designated pSRBD0, was digested with XhoI and ligated with a 1.8 kb XhoI fragment from pJW88 (J.Wieser, unpublished) containing the A.nidulans argB gene to yield pSRBD1 which was used to transform A.nidulans PW1 strain. The fadA^Q204L and fadA^R178C dominant-activating alleles were generated by site-directed mutagenesis as described previously (Yu et al., 1999) and then moved into pSH96 (Wieser and Adams, 1995) to give pJY.26SH and pJY.27SH, respectively.

To isolate mutant alleles of sfaD, we constructed genomic libraries using a plasmid vector that contained the A.nidulans argB⁺ gene (pPK1; Wieser and Adams, 1995). The libraries were constructed by cutting genomic DNA from the different sfaD strains with PstI, HindIII, XhoI, XbaI and SacII and then ligated to PstI-cut pPK1. To test for dominance or recessives of the sfaD allele mutations a flbA^- strain (RJY98.22) was transformed with pPK1 plasmids containing inserts (3.2 kb PstI fragments) encoding the different sfaD alleles.

Photomicrographs of hyphal development were taken using an Olympus BH2 compound microscope and differential interference contrast optics. All other microscopy was carried out using an Olympus SZ-11 stereo microscope and transmitted light.

We thank our colleagues in the laboratory for many helpful suggestions. This work was supported by National Institutes of Health grant GM45252 grant to T.H.A. and Hellmuth Hertz Foundation and the Swedish Institute postdoctoral fellowship to S.R.

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