Genetic Involvement of a cAMP-Dependent Protein Kinase in a G Protein Signaling Pathway Regulating Morphological and Chemical Transitions in Aspergillus nidulans

Corresponding author: Nancy P. Keller, 882 Russell Labs, Department of Plant Pathology, 1630 Linden Dr., University of Wisconsin, Madison, WI 53706., npk{at}plantpath.wisc.edu (E-mail)

Communicating editor: M. E. ZOLAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the filamentous fungus Aspergillus nidulans, a heterotrimeric G protein -subunit and an RGS domain protein, encoded by fadA and flbA, respectively, regulate production of the carcinogenic metabolite sterigmatocystin (ST) and asexual spores (i.e., conidia). We investigated the genetic involvement of the cAMP-dependent protein kinase catalytic subunit (PkaA), a potential downstream target of FadA activity, in ST production and conidiation. Relative to wild type, sporulation was decreased in the pkaA overexpression strain but was not totally absent, as occurs in flbA or fadA^G42R (fadA-dominant active) strains. Deletion of pkaA resulted in a hyper-conidiating strain with limited radial growth. This phenotype was epistatic to mutation in flbA or fadA; the double mutants pkaA; flbA and pkaA; fadA^G42R recovered sporulation and their radial growth was severely restricted. PkaA overexpression also negatively regulated AflR, the ST biosynthesis-specific transcription factor, both transcriptionally and post-transcriptionally. Deletion of pkaA restored ST production in the flbA background but not in the fadA^G42R background. These data provide genetic evidence that the FlbA/FadA signaling pathway regulating ST production and morphological development is partially mediated through PkaA.

THE polyketide sterigmatocystin (ST) is a carcinogenic secondary metabolite produced by Aspergillus nidulans. The biosynthetic genes and the gene encoding the pathway-specific transcription factor of this metabolic pathway have been cloned and functionally analyzed (BROWN et al. 1996 ; FERNANDES et al. 1998 ). Interestingly, ST biosynthesis is genetically linked with asexual development (i.e., conidiation) in this fungus (HICKS et al. 1997 ). Two of the genes required for ST production and conidiation, flbA and fadA, are involved in a G protein signaling pathway. FlbA contains an RGS (regulator of G protein signaling) domain in its C-terminal region, a conserved domain that is implicated in negatively regulating G protein-mediated signaling pathways through enhancing the endogenous GTPase activity in -subunits of G proteins (DE VRIES and GIST FARQUHAR 1999 ). FadA, which is similar to the inhibitory -subunit of heterotrimeric G proteins (G_(i)) of other organisms, appears to be a target of FlbA (YU et al. 1996B ). aflR, a gene encoding a ST biosynthesis pathway-specific transcription factor, and brlA, encoding a conidiation-specific transcription factor, require FlbA and FadA for their normal expression. When flbA is eliminated, or FadA is activated, aflR and brlA expression and subsequent ST production and conidiation are inhibited (YU et al. 1996B ; HICKS et al. 1997 ). On the other hand, overexpression of flbA or the presence of a dominant negative allele of fadA (fadA^d-) allows unscheduled and early expression of these transcription factors and subsequent ST and conidia production (HICKS et al. 1997 ). However, the genetic pathway connecting these four genes (or their products) has not been elucidated.

A candidate pathway genetically linking FadA activity to regulation of BrlA and AflR would be the adenylyl cyclase/cAMP/cAMP-dependent protein kinase (PKA) cascade. Activity of adenylyl cyclase, which synthesizes the intracellular messenger cAMP, has been shown to be regulated by -subunits of heterotrimeric G proteins (G), which either stimulate (G_(s)) or inhibit (G_(i)) adenylyl cyclase activity in a number of diverse organisms (DOHLMAN et al. 1991 ; QUAN et al. 1991 ; ISSHIKI et al. 1992 ; CHEN et al. 1996 ; KORSWAGEN et al. 1998 ; IVEY et al. 1999 ). When adenylyl cyclase is active, the intracellular concentration of the second messenger cAMP is elevated. cAMP activates PKA, which is composed of two catalytic subunits (PKAC) and two regulatory subunits, by binding to the regulatory subunits, which results in release of the active catalytic subunits. A connection between G-proteins, adenylyl cyclase, and PKA activation has been genetically characterized in Saccharomyces cerevisiae (THEVELEIN and DE WINDE 1999 ) and to some extent in the corn smut fungus Ustilago maydis (KRONSTAD et al. 1998 ) and the rice blast fungus Magnaporthe grisea (ADACHI and HAMER 1998 ).

Multiple PKAC genes have been characterized for two fungi, U. maydis and S. cerevisiae. U. maydis contains two PKAC genes, adr1 and uka1. Inactivation of adr1 resulted in constitutive filamentous growth and avirulence whereas deletion of uka1 did not affect fungal morphology or pathogenicity (DURRENBERGER et al. 1998 ). S. cerevisiae contains three PKAC proteins, encoded by TPK1, TPK2, and TPK3, with overlapping roles for viability (TODA et al. 1987 ), but recent studies showed that each catalytic subunit has a distinctive function for pseudohyphal growth, iron uptake, and respiration (ROBERTSON and FINK 1998 ; ROBERTSON et al. 2000 ). In both of these examples, environmental signals leading to developmental processes were transduced through PKA via upstream components including receptors, G proteins, and/or adenylyl cyclase.

The strongest evidence to support a genetic connection between PKA and other members of signaling pathways in fungi has been through mutagenesis and pathway studies detailed in S. cerevisiae and U. maydis (reviewed in KRONSTAD et al. 1998 ; THEVELEIN and DE WINDE 1999 ). However, biochemical experiments have also supported the existence of a G protein/adenylyl cyclase/cAMP/PKA signaling pathway in pathogenic fungi. For example, inactivation of the adenylyl cyclase gene, which decreases the intracellular concentration of cAMP and presumably concomitant PKA activity, has been associated with aberrations in sporulation, virulence, and mating (GOLD et al. 1994 ; CHOI and DEAN 1997 ). The defects in adenylyl cyclase mutants resembled those of PKA catalytic subunit mutants (MITCHELL and DEAN 1995 ; XU et al. 1997 ; DURRENBERGER et al. 1998 ). Furthermore, exogenous cAMP partially restored wild-type phenotypes in M. grisea and U. maydis G or adenylyl cyclase mutants, thus suggesting that PKA is the downstream target of G protein and/or adenylyl cyclase (GOLD et al. 1994 ; CHOI and DEAN 1997 ; LIU and DEAN 1997 ; REGENFELDER et al. 1997 ). In the human pathogen Cryptococcus neoformens, production of the pathogenicity factor melanin was affected by alterations in cAMP levels (ALSPAUGH et al. 1997 ). This report implies but does not genetically analyze a role for PKA in secondary metabolism.

Considering this accruing evidence that PKA is a member of the G protein signaling pathway in fungi, and furthermore, that this protein plays vital roles in regulating such diverse processes as spore production and, possibly, secondary metabolism in pathogenic fungi, we were interested in examining whether PKA is involved in linking FlbA and FadA activity to conidiation and ST production in the mycotoxigenic fungus A. nidulans. In this study, we demonstrate that a cAMP-dependent protein kinase catalytic subunit, PkaA, mediates FlbA and FadA effects on both asexual development and secondary metabolism in A. nidulans.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Fungal strains and media:
Fungal strains used in this study are listed in Table 1. All strains were maintained on Aspergillus minimal agar medium (MMG: 6.0 g NaNO₃, 0.52 g KCl, 0.52 g MgSO₄ · 7H₂O, 1.52 g KH₂PO₄, 1 ml trace elements [2.2 g ZnSO₄ · 7H₂O, 1.1 g H₃BO₃, 0.5 g MnCl₂ · 4H₂O, 0.5 g FeSO₄ · 7H₂O, 0.16 g CoCl₂ · 5H₂O, 0.16 g CuSO₄ · 5H₂O, (NH₄)₆Mo₇O₂₄ · 4H₂O, 5.0 g Na₄ EDTA in 100 ml distilled H₂O], 10 g glucose, 12.5 g agar, pH 6.5, in 1 liter distilled H₂O) with appropriate supplements (KAFER 1977 ). Glucose was replaced with 100 mM threonine (MMT) as indicated. Agar was not added for liquid medium. For genomic DNA extraction and protoplast preparation, strains were grown on appropriately supplemented liquid minimal medium, and 0.5% yeast extract was added when required. For RNA extraction from double mutant strains, complete medium (CM) was used as described previously (HICKS et al. 1997 ).

Cloning and sequencing of pkaA:
The expressed sequence tag clone n8d03a1, whose nucleotide sequence was highly homologous to those of fungal genes encoding cAMP-dependent protein kinase catalytic subunit, was used to screen an A. nidulans cosmid library. Two overlapping cosmid clones, pWE08C4 and pWE06A11, were selected. A 5.4-kb HindIII fragment of pWE08C4 was cloned into pK19 to produce pKIS1. Restriction mapping and sequencing of pKIS1 revealed that the catalytic core of PkaA lay in the middle of the insert within a 1.5-kb BamHI internal fragment. The nucleotide sequence of the pkaA gene was determined on both strands by the dideoxy chain termination method (SANGER et al. 1977 ).

Creation of pkaA deletion and pkaA overexpression strains:
The 5.4-kb HindIII fragment from pKIS1 was placed into a modified pBluescript vector (pKIS16) in which the BamHI site had been removed by ligating the blunt-ended BamHI/SacII sites together. The resulting plasmid, pKIS17, was then digested with BamHI to release the 1.5-kb fragment, which was replaced with a 1.8-kb BamHI fragment from pJYargB2 containing the A. nidulans argB gene. The resulting plasmid pKIS18, which eliminated most of the deduced PkaA, was used to transform A. nidulans strain FGSC851 to arginine prototrophy. The deletion in the transformant TKIS18.11 was confirmed by genomic Southern analysis (data not shown). The 2.3-kb SphI fragment containing the entire coding region of the pkaA gene was ligated into the SphI site of pBN55 (LEE and ADAMS 1994 ), which contains a 1.75-kb fragment containing the 5' portion of the trpC gene, which can complement trpC801 mutation, and alcA(p), a promoter of the alcohol dehydrogenase gene. The resulting plasmid, pKIS20, has the pkaA fragment fused to the alcA(p), which can be activated by supplementing media with specific carbon sources such as threonine or ethanol. pKIS20 was introduced into FGSC237, and a tryptophan prototroph containing the alcA(p)::pkaA construct was isolated and named TKIS20.1.

PKA activity assay:
Two hundred and fifty milliliters of liquid minimal media was inoculated with 1 x 10⁶ conidia/ml and incubated for 14 hr at 37° and 300 rpm. The mycelium was collected, washed, divided into equal parts, and transferred to flasks with 50 ml minimal media with 100 mM L-threonine as the sole carbon source or 50 ml minimal media with 1% glucose as the sole carbon source. Samples were incubated for an additional 12 hr at 300 rpm and 37° before harvesting, washing with phosphate buffered saline (0.2 g/liter KCl, 8.0 g/liter NaCl, 0.2 g/liter KH₂PO₄, 1.15 g/liter Na₂HPO₄), freezing in liquid nitrogen, and lyophilizing. The lyophilized mycelia were ground and mixed with phosphate buffer [50 mM Na₂HPO₄ (pH 7.0), 10 mM EDTA, 10 mM ß-mercaptoethanol, 0.1% sarcosyl, 0.1% Triton X-100, 0.1 mM phenylmethyl-sulfonyl fluoride]. After centrifugation at 14,000 x g for 20 min, the supernatants were collected and used to determine protein concentration (BRADFORD 1976 ) and were adjusted to 1.5 µg protein/µl. The assay for PKA activity was performed with SignaTECT cAMP-dependent protein kinase assay system (Promega, Madison, WI) according to the manufacturer's directions.

Creation of double mutant strains:
RKIS3.6 (pkaA; flbA) was obtained from a sexual cross between TKIS18.11 and RJH047 (Table 1). The pkaA and flbA alleles were confirmed by genomic Southern hybridization. pKIS15 was constructed by introducing a 3.2-kb PstI fragment of pJY8P2 containing the fadA dominant activating allele, fadA^G42R, into pSM3, which contains pyroA as a selectable marker. A. nidulans strain RKIS3.15, a progeny of the sexual cross between TKIS18.11 and RJH047, was transformed with pKIS15 to introduce fadA^G42R into the pkaA background. Diploid strains DKIS2 and DKIS3 were constructed with haploid strains TKIS20.1 and RJH079 or RKIS1 and RJH079.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The A. nidulans pkaA gene encodes the catalytic subunit of a PKA protein:
Sequence analysis of the putative cAMP-dependent protein kinase catalytic subunit gene (pkaA) revealed a 1416-bp open reading frame encoding a 472-amino-acid protein with a predicted molecular mass of 53.8 kD interrupted by three introns, 58 bp, 52 bp, and 51 bp in length (GenBank accession no. AF262987). Southern analysis showed that A. nidulans has only one copy of pkaA in the genome (data not shown). The catalytic core of protein kinase, as defined by HANKS and HUNTER 1995 , appears to be highly conserved in this gene. From amino acids 168 to 173 there is a consensus sequence for an ATP-binding site, GXGXXG, followed eight residues later by K (lysine), a motif found in protein kinases of other organisms (HUNTER and COOPER 1985 ). A GenBank search showed that PkaA is similar (32% identity) to numerous eukaryotic genes, including those of yeast, which encode PKACs. This was particularly true in comparing A. nidulans PkaA to that of other filamentous fungi (52–70% identity). Alignment of the amino acid sequence showed that all the fungal species examined have a highly conserved catalytic core, but the N termini of PKACs is quite variable except for the first 15 amino acids (data not shown).

PKA activity is increased in a pkaA overexpression strain and is decreased but not eliminated in a pkaA deletion strain:
To assess the function of pkaA and its protein product, strains were created in which the gene was deleted and overexpressed. pkaA patterns are shown in Fig 1A. In wild-type strains, pkaA appears to be expressed at basal levels over the time points tested in both glucose and threonine media. The overexpression strain TKIS20.1, which contains both a wild-type allele and a copy of pkaA fused to the inducible promoter alcA(p), showed much higher accumulation of pkaA in alcA-inducing medium (MMT). In addition, even in glucose media, slightly higher pkaA transcripts were detected in the overexpression strain as compared to the wild-type strain. This result indicates that alcA(p) is activated to a low level in 1% glucose. This was confirmed by probing the same RNA blot with the alcA gene (data not shown). As expected, no transcript was observed in either medium in the pkaA deletion strain TKIS18.11. Next we determined PKA activity in these three strains (Fig 2). TKIS20.1 showed much higher PKA activity in threonine medium compared to wild type. In TKIS18.11, PKA activity in both glucose and threonine was much lower than in the wild-type strain, but was still significantly greater than the negative control of boiled protein (data not shown).

View larger version (75K): In this window In a new window Download PPT slide   Figure 1. pkaA mutants affect pkaA, stcU, and brlA expression. Total RNA was isolated from RKIS1 (wild type), TKIS20.1 (alcA(p)::pkaA), and TKIS18.11 (pkaA) cultures 0, 12, 24, and 48 hr after transferring from MMG to MMG or from MMG to MMT (an alcA-inducing medium). A 1.5-kb BamHI fragment of pKIS17 was used as a pkaA-specific probe (A). A 4.5-kb SalI fragment of pTA111 (ADAMS et al. 1988 ) was used as a brlA-specific probe (B). A 1.3-kb EcoRV-XhoI fragment of pAHK25 (BROWN et al. 1996 ) was used as a aflR-specific probe (C). A 0.75-kb SstII-SmaI fragment of pRB7 (YU et al. 1996A ) was used as a stcU-specific probe (D). Equal loading of total RNA was evaluated by ethidium bromide staining (E).

View larger version (28K): In this window In a new window Download PPT slide   Figure 2. PKA activity in pkaA mutant strains. Total protein was prepared from RKIS1 (wild type), TKIS20.1 (alcA(p)::pkaA), and TKIS18.11 (pkaA) from cultures 0 and 12 hr after transferring from MMG to MMG or MMG to MMT (alcA-inducing condition). Standard error bars are shown for three replicates.

PkaA inactivation and overexpression alter morphological development in A. nidulans:
To examine if PkaA had an effect on differentiation in A. nidulans, morphological development of strains containing pkaA and alcA(p)::pkaA alleles were compared and contrasted to wild type. If, as hypothesized, FadA signaling is mediated through PkaA, we would expect that the phenotype of pkaA mutants would mimic certain fadA and flbA mutants. For example, if FadA is a G_(i)-subunit resulting in decreased PKA activity, then the pkaA might resemble a FadA activating mutant such as represented by a loss-of-function flbA allele or a fadA^G42R allele. On the other hand, if FadA is a G_(s)-subunit, then one would expect that the enhanced PkaA activity of the pkaA overexpression strain would mimic the phenotype associated with flbA or fadA^G42R. flbA or fadA^G42R mutants, which result in near-constitutive G-signaling, exhibit extreme aerial hyphal growth with a fluffy appearance and autolyse as colonies mature. They also are unable to produce conidia on MMG.

We examined the phenotype of the pkaA overexpression strain by evaluating growth on glucose agar medium, MMG (non-inducing), and threonine agar medium, MMT (inducing), and by comparing growth habits to that of an isogenic wild-type strain grown in the same conditions (Fig 3). There were considerably more aerial mycelia produced by TKIS20.1 when grown on MMT giving the colony a fluffy phenotype (Fig 3). In addition, TKIS20.1 also showed a significant reduction in the density of conidia per unit area when grown on MMT as compared to wild type (84% reduction, Table 2). These growth and sporulation habits, while not as severe as those seen in the flbA or fadA^G42R phenotype, did nevertheless resemble this phenotype.

View larger version (82K): In this window In a new window Download PPT slide   Figure 3. Growth of mutant strains on solid media. Fungal strains, RKIS1 (wild type, left) and TKIS20.1 (alcA(p)::pkaA, right) were grown on either MMG or MMT solid media for 5 days at 37° after inoculation.

The growth habits of the pkaA strain on MMG solid medium also differed from the growth habits of the wild type in two key aspects. First, radial growth rate was reduced by 85% in TKIS18.11 (pkaA) compared to the wild-type strain (Table 3). Second, conidial production per unit area of the pkaA strain was significantly higher than wild type (1.5-fold increase). The phenotype of TKIS18.11 showed some similarities to that of A. nidulans strains containing a fadA^G203R allele, a dominant-interfering mutation that generates a G-protein constitutively bound to guanosine diphosphate (YU et al. 1996B ).

Morphological observations were also made on growth habits in submerged culture. Wild-type strains of A. nidulans normally do not sporulate in submerged glucose culture, and, as shown in Fig 4, pkaA and alcA(p)::pkaA strains also did not sporulate under these conditions at the time points tested. In this instance, the pkaA strain did not resemble the fadA^G203R strain as the latter does sporulate in submerged culture (HICKS et al. 1997 ). Microscopic examination revealed, however, that the pkaA strain had a thinner hyphal diameter than the other two strains (Fig 4C). When grown in threonine media, TKIS20.1 did not develop conidia (Fig 4E) whereas both the wild type and TKIS18.11 produced conidia from conidiophores (Fig 4D and Fig F). Both the wild type and the deletion strain sporulated within 12 hr after shifting from glucose to threonine medium. Notably, the pkaA strain sporulated with little mycelial growth in contrast to wild type, which produced copious amounts of mycelia. Expression of brlA, which is required for conidiophore development, was not detected in the pkaA overexpression strain, but was detected both in the wild type and to a higher level in the pkaA deletion strain grown in threonine medium (Fig 1B). This was consistent with the results of observations shown in Fig 4D–F. In addition to producing conidiophores, the pkaA strain also formed many large spherical cells in threonine medium (Fig 4F). The fact that the overexpression strain did not sporulate in submerged threonine-inducing media, unlike wild-type and pkaA strains, and that it exhibited reduced conidiophore formation on MMT agar medium (Table 2 and Fig 3) demonstrates that PKA activity inhibits asexual development, a phenotype associated with constitutive FadA signaling.

View larger version (114K): In this window In a new window Download PPT slide   Figure 4. Growth of different pkaA mutant strains in submerged cultures. RKIS1 (wild type, A and D), TKIS20.1 (alcA(p)::pkaA, B and E), and TKIS18.11 (pkaA, C and F) were grown in MMG liquid shake cultures at 37° for 24 hr and transferred to MMG (A–C) or MMT (D–F) for an additional 12 hr at 37°. S, asexual spore.

PkaA mediates FadA and FlbA growth signaling:
To determine genetically if PkaA was involved in mediating the FadA signal in asexual development, we introduced flbA and fadA mutant alleles into the pkaA background to examine epistatic relationships among these genes. Morphological characteristics of these strains are summarized in Table 3. A striking phenotype of strains carrying either loss-of-function flbA (flbA) or dominant positive fadA alleles (fadA^G42R) is the inability to produce conidiophores, abundant aerial mycelia, and autolysing of the mature colony (YU et al. 1996B ). As described in the previous section, the distinct phenotype of pkaA strains is the severe restriction of the radial growth rate and an increase in the number of conidia/area. Both double mutant strains, pkaA; fadA^G42R and pkaA; flbA, exhibited the restricted radial growth phenotype of a pkaA strain and an intermediate sporulation phenotype compared to the single mutant strains (Table 3). Thus in A. nidulans, colony expansion seems to be largely contributed by PkaA activity, but normal asexual sporulation may require not only inactivation of PkaA but also active FlbA and subsequent inactivation of FadA. Interestingly, the pkaA; flbA strain but not the pkaA; fadA^G42R strain developed aerial hyphae. Additionally, unlike the flbA or fadA^G42R single mutant strains, neither the pkaA; flbA nor the pkaA; fadA^G42R strain autolysed. Mycelial autolysis may require both activated FadA and PkaA in A. nidulans.

aflR and stc gene expression and ST production is repressed in pkaA overexpression strains:
Production of the mycotoxin ST has been genetically linked to conidiation through FadA signaling (HICKS et al. 1997 ). It was therefore of interest to determine whether the pkaA mutant strains also were affected in ST production. Because ST production is heralded by the appearance of ST gene cluster transcripts (BUTCHKO et al. 1999 ), we examined expression of aflR, the ST pathway-specific transcription factor, and stcU, one of the biosynthetic genes involved in ST biosynthesis (BROWN et al. 1996 ; FERNANDES et al. 1998 ), in pkaA mutant strains. In the wild-type strain, both aflR and stcU transcripts were detected at 36 and 48 hr after shifting from MMG to MMG and at 24 hr when shifted from MMG to MMT (Fig 1C and Fig D). In the pkaA strain, expression of both genes was delayed in both media and decreased in intensity in MMG medium. Neither aflR nor stcU transcript was detected in the pkaA overexpression strain, whether grown in MMG or MMT media. This suggested that even the small increase of PkaA activity observed in 1% glucose medium was sufficient to inhibit ST production.

PkaA activity regulates aflR not only transcriptionally but also post-transcriptionally:
Because the expression of aflR was shown to be negatively regulated by PkaA activity, we were interested to see if we could remediate this regulation by forced expression of aflR in the pkaA overexpression background. Thus, we constructed diploid strains carrying alcA(p)::aflR and alcA(p)::pkaA alleles or alcA(p)::aflR alone and grew these strains in alcA-inducing and non-inducing conditions. As expected, significant expression of aflR was not observed in MMG medium but was observed in both diploid strains in MMT (Fig 5). However, despite high aflR expression, stcU expression in MMT was detected in only the wild-type pkaA diploid and not in the pkaA overexpression diploid, suggesting that aflR activity is post-transcriptionally regulated by PkaA. Taken together with the results shown in the previous section, we conclude that aflR is negatively regulated both transcriptionally and post-transcriptionally by PkaA activity. At present, the mechanism by which PkaA regulates aflR is not known, but it is interesting to note that the A. nidulans AflR protein contains three putative PKA-specific phosphorylation motifs (data not shown).

View larger version (101K): In this window In a new window Download PPT slide   Figure 5. stc gene expression in the pkaA/aflR overexpression strains. Fungal diploid strains, DKIS2 (alcA(p)::aflR/alcA(p)::pkaA) and DKIS3 (alcA(p)::aflR), were grown in liquid shaking MMG for 14 hr at 37°, transferred to MMG or MMT, and incubated for additional 12 or 24 hr at 37°. (A) pkaA transcript, (B) aflR transcript, (C) stcU transcript; (D) staining of total RNA with ethidium bromide.

Deletion of pkaA remediates the inhibitory effect of a loss-of-function flbA allele on ST production:
Because of poor growth of the double mutant strains in liquid minimal medium, fungal cultures were grown in CM to assay stc expression. In the wild-type strain, both aflR and stcU transcripts were observed at 48 hr and lower expression was seen at 72 hr after inoculation (Fig 6, lanes 1 and 2). In the pkaA strain expression of both genes was slightly less than that of the wild-type strain at both time points (Fig 6, lanes 3 and 4). Both the flbA and fadA^G42R strains showed no expression of either aflR or stcU (Fig 6, lanes 5–8). The pkaA; flbA double mutant strain showed both aflR and stcU transcripts (Fig 6, lanes 9 and 10). In contrast, however, the pkaA; fadA^G42R strain did not show either aflR or stcU transcript in this growth condition (Fig 6, lanes 11 and 12). Analysis of extracts from these strains revealed a corresponding pattern of ST production (data not shown).

View larger version (72K): In this window In a new window Download PPT slide   Figure 6. aflR and stc gene expression in pkaA deletion strains. Fungal strains were grown in liquid stationary culture (CM) and harvested at 48 and 72 hr post-inoculation: lane 1, wild type; lane 2, pkaA; lane 3, flbA; lane 4, fadAG42R; lane 5, pkaA; flbA; lane 6, pkaA; fadAG42R. (A) aflR transcript, (B) stcU transcript; (C) staining of total RNA with ethidium bromide.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In many fungi, a correlation between asexual development (e.g., conidiation) and secondary metabolism has been recognized, but the cellular machinery linking these two processes is largely unclear. We have initiated an investigation of the pathways connecting sporulation and secondary metabolism in the genetic model system A. nidulans. At least two genes, flbA and fadA, have been identified as common factors in regulating conidiation and ST production by controlling expression of the specific transcription factor genes brlA (conidiation) and aflR (ST production; YU et al. 1996B ; HICKS et al. 1997 ). FlbA and FadA are members of a G protein signal transduction pathway and the molecular mechanisms connecting these two proteins with aflR and brlA activity remain unknown. One logical candidate for transmitting a signal from FlbA and FadA to aflR and brlA would be cAMP-dependent PKA.

To determine if PKA could be a component in this transduction pathway, we cloned and characterized the gene encoding the cAMP-dependent protein kinase catalytic subunit, pkaA. Amino acid analysis of PkaA shows it to contain the highly conserved catalytic core common to numerous eukaryote PKAC proteins. The N-terminal domain shows little similarity to any of these proteins with the exception of the first dozen amino acids, which were similar to those of other filamentous fungal PKACs (data not shown). Little is known about the function of the N-terminal region; in Dictyostelium discoideum, deletion of this portion showed no phenotype (ETCHEBEHERE et al. 1997 ). Comparison of the three yeast PKAC proteins, however, suggests that the variation in their N-terminal domain contributes to the differences in their function (ROBERTSON and FINK 1998 ). We have seen an inhibitory effect on ST production and altered morphological development by adding an extra copy of the A. nidulans N-terminal region (K. SHIMIZU and N. P. KELLER, unpublished results).

Expression of pkaA was detected at nearly the same level in all the time points tested in this study in the wild-type strain, suggesting that pkaA is expressed constitutively. In other fungal species such as C. trifolii, Ct-PKAC expression also has been detected at all time points examined (YANG and DICKMAN 1999 ). Basal level expression of a gene sometimes indicates an absolute requirement for the activity of the gene product in the cell. However, we found that while deletion of pkaA did exhibit a severe phenotype, it was not lethal in A. nidulans. This may be because A. nidulans possesses another PKAC protein as indicated by the low but measurable level of PKA activity in the pkaA strain. This would not be without precedent because both U. maydis and S. cerevisiae have more than one gene encoding PKAC (TODA et al. 1987 ; DURRENBERGER et al. 1998 ). In these two fungi the different PKAC proteins appear to play different but overlapping roles in the cell (DURRENBERGER et al. 1998 ; ROBERTSON and FINK 1998 ; ROBERTSON et al. 2000 ). In S. cerevisiae lethality is exhibited only when all three genes encoding PKAC are disabled (TODA et al. 1987 ).

Examination of pkaA deletion and overexpression strains indicated a role for PkaA in several aspects of Aspergillus development. Consistent observations included the complete inhibition of stcU expression and ST production and partial inhibition of asexual development in pkaA overexpression strains and the reduced radial growth and increased sporulation of pkaA strains. Interestingly, the data shown here suggest that the ST biosynthesis-specific transcription factor AflR is transcriptionally and post-transcriptionally regulated by PkaA activity, a hypothesis we are presently examining in our laboratory. These results strongly support a major role of PkaA activity in regulating conidiation, vegetative growth, and secondary metabolism in this fungus. Notably, the phenotype of pkaA overexpression strains was reminiscent of loss-of-function FlbA and FadA constitutive activating mutants of A. nidulans and suggested that, as in other organisms, RGS/G protein signaling is mediated in part through the cAMP/PkaA cascade. This also implies that FadA may function as a G_(s)-subunit despite its sequence similarity to mammalian G_(i)-subunits. Interestingly, most fungal G-genes cloned to date show this same sequence similarity to G_(i)-subunits but have been found to more likely function as activators of adenylyl cyclase, while that of Cryphonectria parasitica seems to work as G_(i) (ISSHIKI et al. 1992 ; CHEN et al. 1996 ; SAVINON-TEJEDA et al. 1996 ; ALSPAUGH et al. 1997 ; LIU and DEAN 1997 ; KRUGER et al. 1998 ; IVEY et al. 1999 ).

The conclusion that FadA activates PkaA is also supported by examining the phenotypes of double mutants with regard to several key differentiation processes. One of the most dramatic effects of constitutive activation of G protein signaling in A. nidulans is the inhibition of asexual sporulation. We found that introduction of the pkaA allele into flbA and fadA^G42R backgrounds restored conidiation; this provides strong evidence that FadA inhibition of asexual development is mediated through PkaA. However, remediation was not to wild-type levels (pkaA; flbA showing 15% and pkaA; fadA^G42R showing 40% sporulation levels of wild type). Previous studies (YU et al. 1996B ; HICKS et al. 1997 ) suggested that FlbA has some additional function(s) in regulating sporulation other than through modification of FadA activity. Therefore, one explanation as to why sporulation was not fully restored in the pkaA; flbA strain is that pkaA cannot complement a FadA-independent role for FlbA. Sporulation in the pkaA; fadA^G42R strain was statistically greater than in the pkaA; flbA strain but still less than that of wild type. This indicates a PkaA-independent role for FadA in asexual sporulation. As mentioned earlier, other fungi possess more than one PKAC protein and as our data provide evidence for more than one PKAC in A. nidulans (Fig 2), we speculate that FadA activity may also negatively impact sporulation through another PKAC or, although untested in this work, perhaps mitogen-activated protein kinase (MAPK) cascade. A model for this complex regulation of asexual development is presented in Fig 7.

View larger version (24K): In this window In a new window Download PPT slide   Figure 7. Proposed model for a signal transduction pathway regulating both ST production and asexual development. The model is described in the text. Solid lines indicate genetically determined steps and dashed lines hypothesized steps. PkaA may phosphorylate an unidentified transcription factor, which triggers transcription of aflR, and the transcribed aflR is also regulated by PkaA activity. Regulation of morphological development by FlbA and FadA is only partially mediated through PkaA.

The genetic linkage of asexual sporulation and ST production was evident from examination of the various pkaA mutants. The inhibitory effect of overexpression of pkaA on aflR expression and ST biosynthesis was identical to the inhibitory effects of flbA and fadA^G42R alleles on aflR expression. This suggested that FlbA and FadA regulation of ST production is mediated through PkaA, leading therefore to the prediction that introducing pkaA into flbA and fadA^G42R backgrounds would restore ST production. Interestingly, this was the case for the pkaA; flbA strain but not the pkaA; fadA^G42R strain under the conditions tested. The inability to detect ST or stc expression in the pkaA; fadA^G42R strain suggests that FadA also has a PkaA-independent role in inhibiting ST production (Fig 7).

PkaA activity also affected several aspects of Aspergillus vegetative growth including radial expansion rate, aerial mycelium formation, and mycelial autolysis. Strains containing flbA and fadA^G42R alleles produce extensive aerial mycelium and have a habit of autolysing as the colony matures. pkaA overexpression strains exhibited the former characteristic but not the latter. The radial expansion rate appears tightly linked to PkaA activity because all strains carrying the pkaA allele (pkaA, pkaA; flbA, and pkaA; fadA^G42R) showed a similarly restricted expansion rate. Aerial mycelium formation, although enhanced by pkaA overexpression (Fig 3), was not as prolific as seen in flbA or fadA^G42R strains and, furthermore, was not fully dependent on PkaA activity as judged by the fact that aerial mycelium formation was not eliminated in the pkaA; flbA strain. Mycelial autolysis appears to require PkaA activity because deletion of pkaA eliminated the autolytic habit of the flbA and fadA^G42R alleles as observed in pkaA; flbA and pkaA; fadA^G42R strains. The fact that overexpression of pkaA did not result in mycelial autolysis suggests that autolysis requires both activation of FadA and subsequent increased PkaA activity.

Consideration of all data presented in this article leads us to conclude that FlbA/FadA signaling of asexual sporulation, ST production, and vegetative growth is mediated only in part through a cAMP/PkaA cascade (Fig 7). Obviously there must be other cellular mechanisms impacting all of these differentiation processes. In other fungi, transduction pathways are complex and can involve interactions between ß- and -subunits of heterotrimeric G proteins, RAS proteins, MAPK cascades, and protein kinase C pathways (BANUETT 1998 ; RUPP et al. 1999 ; THEVELEIN and DE WINDE 1999 ). Two other pertinent A. nidulans genes that have been shown to be involved in sporulation and colony formation are rasA, encoding a RAS protein (SOM and KOLAPARTHI 1994 ) and sfaD, encoding a ß-subunit of a heterotrimeric G protein (Gß; ROSEN et al. 1999 ). In submerged culture conditions, deletion of sfaD exhibits conidiation, a similar phenotype to a fadA^G203R strain (ROSEN et al. 1999 ), but unlike a fadA^G203R strain, spore production is decreased on agar growth medium (K. SHIMIZU and N. P. KELLER, unpublished results). Dominant-acting mutations in rasA (RAS) result in developmental defects similar to overexpression of pkaA (SOM and KOLAPARTHI 1994 ; K. SHIMIZU and N. P. KELLER, unpublished results). In addition to these proteins, A. nidulans likely contains at least two additional G-subunits (J. YU, personal communication) as well as a possible additional PkaA as suggested by the data in Fig 2. One current goal in our laboratory is to determine if these different members of signal transduction pathways converge to impact morphogenesis and ST production in A. nidulans.

	ACKNOWLEDGMENTS

We are grateful to Tom Fulton for stimulating discussions, to Heather Wilkinson for statistical analysis, to Julie Hicks for providing fungal strains, and to Sungchur Sim for experimental assistance. This work was supported by a Merck & Co. Study Agreement to K.S. and N.P.K.

Manuscript received August 8, 2000; Accepted for publication October 24, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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