Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Corresponding author: Cletus A. D'Souza, Rm. 320, CARL Bldg., Box 3546, Department of Genetics, Duke University Medical Center, Durham, NC 27710., dsouz003{at}mc.duke.edu (E-mail)

Communicating editor: J. J. LOROS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We showed previously that a fluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of fluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from flbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

FORMATION of organs in multicellular eukaryotes is a complex process involving a multitude of sensory pathways that function in perceiving signals from the extracellular environment. Cells are then directed to form higher order structures that perform specific functions. The genetically tractable filamentous fungus Aspergillus nidulans serves as a model organism for the study of complex multicellular development in fungi and other eukaryotes. The asexual phase in the life cycle of this fungus involves the formation of multicellular structures called conidiophores that produce chains of uninucleate spores called conidia (TIMBERLAKE 1990 ). Initiation of conidiation is a programmed event that is genetically determined and occurs at a precisely scheduled time in the A. nidulans life cycle (AXELROD 1972 ). The primary activator of conidiation-specific genes is BrlA, a C₂H₂ zinc-finger DNA-binding protein (ADAMS et al. 1988 ; ADAMS and TIMBERLAKE 1990 ; CHANG and TIMBERLAKE 1992 ). The isolation of conidiation mutants has facilitated the identification of several genes required for brlA expression and the programmed induction of conidiophore development, namely flbA, fluG, flbB, flbC, flbD, and flbE (LEE and ADAMS 1994A ; WIESER et al. 1994 ; Fig 4). These mutants have a predominantly fluffy phenotype with conidiation being impaired to varying extents.

View larger version (87K): In this window In a new window Download PPT slide   Figure 1. (A) Development in fluG strains bearing alcA(p)::fluG fusions. Wild-type (TTA11), fluG (TTA127.4), and fluG strains bearing alcA(p) expression constructs with different portions of the fluG gene (indicated in parentheses) were grown in alcA(p)-repressing liquid minimal medium (glucose) for 14 hr at 37° and then shifted to alcA (p)-inducing liquid minimal medium (threonine). Micrographs were taken 24 hr after alcA (p) induction. Conidiophore development (indicated by arrowheads) is observed only in strains overexpressing the entire fluG(1–865; RCD45) and the C-terminal fluG(387–865; RCD47.18) indicating that FluG(387–865) is necessary and sufficient for FluG function. The wild-type strain and fluG strains do not sporulate under these conditions. (B) Complementation of the fluG phenotype in strains bearing plasmids with different portions of fluG under the control of its own promoter introduced at the argB locus. Sporulation (seen as green color of spores in the center of the colony) is observed in fluG + fluG(387–865) strain (TCD13)on solid minimal medium with supplements. Also shown are the fluG and fluG + fluG(1–865; TCD18) strains. Complementation of fluG by fluG(387–865) further confirms the sufficiency of fluG(387–865) for FluG function.

View larger version (112K): In this window In a new window Download PPT slide   Figure 2. (A) Morphology of dsgA1 mutant strains and ability of the dsgA1 mutation to suppress conidiation defects of fluG, flbA, and fadAQ204L mutations. Strains grown on supplemented minimal agar medium for 2 days at 37°. On the left is a plate indicating suppression of fluG by the dsgA1 mutation as seen from conidiation of the strains shown (indicated by the yellow or green color of spores in the colony). The fluG/fluG; dsgA1/+ isolated by NQO mutagenesis of the corresponding homozygous fluG strain (fluffy aconidial) can be seen to sporulate under these conditions, indicating dominant suppression of the fluG mutation. The plate on the right demonstrates the ability of dsgA1 to suppress the flbA and fadAQ204L fluffy autolysis phenotypes. (B) Conidiophore formation in dsgA1 mutant strains grown in liquid shake culture. A total of 5 x 105 conidia/ml from wild type (TTA11), fluG (TTA127.4), fluG; dsgA1 (HMDCD1.4), dsgA1 (HDCD3.8), flbA; dsgA1 (HMDCD4.26), and fadAQ204L; dsgA1 (TJYPK26.15) were inoculated into 100 ml of supplemented liquid minimal medium (KAFER 1977 ) incubated at 37° in a shaker set at 300 rpm. Strains fluG; dsgA1 and dsgA1 were also grown in liquid minimal medium supplemented with 2.0 g/liter of yeast extract. Following 16 hr of growth micrographs were taken of hyphae to observe formation of conidiophores (indicated by arrowheads).

View larger version (71K): In this window In a new window Download PPT slide   Figure 3. dsgA1 does not bypass the requirement of fluG in stcU gene expression. Total RNA was isolated from 1- to 4-day-old liquid stationary cultures of WT (TTA11), fluG (TTA127.4), fluG; dsgA1 (HMDCD1.4), and dsgA1 (HDCD3.8). Equal loading of the RNA was determined by ethidium bromide staining of the gel (bottom). The RNA was hybridized to a stcU-specific radiolabeled probe that detected a 0.9-kbp transcript. No signal was observed for the fluG and fluG; dsgA1 strains, indicating that dsgA1 does not bypass the requirement for fluG in stcU gene expression.

View larger version (17K): In this window In a new window Download PPT slide   Figure 4. Model for the role of DsgA in the asexual sporulation pathway. On solid medium, irrespective of nutrient status or osmolarity, the presence of air induces the FluG-dependent asexual sporulation pathway, which is in competition with the vegetative growth pathway promoted by FadA. FadA also negatively regulates the ST biosynthesis pathway. FluG stimulates FlbA to inactivate FadA but also has a specific role in the asexual sporulation pathway, acting either directly or via FlbA. A role for DsgA as a receptor of the FluG-generated signal or an early component of the signaling pathway leading to activation of brlA is proposed. In the absence of FluG, sporulation then becomes dependent on nutrient status. In liquid media with optimal growth conditions, the growth pathway prevails as the FluG signal cannot compete with the FadA signal. Under stressful conditions (nutrient limitation, osmolarity), FadA signaling is repressed as the FluG pathway is stimulated.

The aconidial phenotype of fluG mutants can be partially rescued in at least two ways. First, fluG mutants can be induced to sporulate by growth on the surface of nutrient-limited media, suggesting that in the absence of fluG conidiation can occur in response to nutritional stress (ADAMS et al. 1992 ). Such stress-induced conidiation may also occur in wild-type strains but is masked by the overwhelming fluG-dependent response. Second, fluG mutants can also be induced to conidiate when grown in proximity to a wild-type strain or strains with mutations in different sporulation genes (LEE and ADAMS 1994A ). This result has led to the proposal that fluG is required for the production of an extracellular factor(s) needed to initiate the developmental pathway leading to conidiation. The mechanism by which FluG activates extracellular factor production remains largely unknown. FluG shares significant sequence similarity with type I glutamine synthetases found in prokaryotes (LEE and ADAMS 1994A ), although FluG-like genes have also recently been found in higher plants (MATHIS et al. 1999 , MATHIS et al. 2000 ).

Overproduction of FluG can drive conidiation in liquid-submerged culture, a condition that is normally repressive for wild-type conidiation (LEE and ADAMS 1995 ). This ability of FluG to initiate conidiation is dependent on the function of FlbA (LEE and ADAMS 1994B , LEE and ADAMS 1995 ), an RGS (regulator of G-protein signaling) domain protein (DE VRIES et al. 1995 ; DRUEY et al. 1996 ; KOELLE and HORVITZ 1996 ; YU et al. 1996 ). RGS proteins stimulate the intrinsic GTPase activity of specific heterotrimeric G-protein -subunits, converting them to the inactive GDP-bound state (BERMAN et al. 1996 ; WATSON et al. 1996 ), and have been implicated in negatively regulating G-protein-mediated signaling pathways (DIETZEL and KURJAN 1987 ; DE VRIES et al. 1995 ; DOHLMAN et al. 1995 ; DRUEY et al. 1996 ; KOELLE and HORVITZ 1996 ; YU et al. 1996 ). FlbA is proposed to inactivate the -subunit of a heterotrimeric G-protein that negatively regulates conidiation, encoded by the fadA (fluffy autolytic dominant) gene (YU et al. 1996 ). Dominant activating mutations in fadA that are predicted to eliminate GTPase activity and lock the protein in a GTP-bound state, like flbA loss-of-function mutations, result in an aconidial phenotype that is characterized by proliferative mycelial growth followed by colony autolysis. A null mutation in fadA is able to suppress the need for flbA in sporulation but does not bypass the need for fluG, indicating that FluG has a specific function in development that is distinct from inhibition of the FadA signaling pathway and presumably involves stimulating products of the flbB, flbC, flbD, flbE, and brlA genes (LEE and ADAMS 1995 ).

Here we describe two approaches to more precisely define the role of FluG in asexual development. First, we have delimited the region of FluG that is required for its activity and show that the glutamine synthetase I (GSI)-like C-terminal half of FluG is sufficient to induce conidiation in this fungus. Second, we describe a genetic screen for dominant suppressors of a fluG mutation (dsg) aimed at identifying components of the conidiation signaling pathway that act downstream of FluG. We reasoned that use of a diploid for the mutant screen would preclude isolation of recessive mutations that cause conidiation as a result of growth inhibition, thereby facilitating the isolation of dominant mutations that would specifically identify activators of the conidiation pathway. Indeed, our results indicate that the mutation dsgA1 may represent a component that is specifically involved in A. nidulans conidiophore development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A. nidulans strains, growth conditions, and genetic analysis:
The strains used in this study are listed in Table 1. Standard A. nidulans genetic (PONTECORVO et al. 1953 ; CLUTTERBUCK 1974 ) and transformation protocols (YELTON et al. 1984 ; MILLER et al. 1985 ) were used. The alcA(p)::fluG transformant strains were generated by integration of plasmid sequences at the trpC locus as described previously (LEE and ADAMS 1995 ). Presence of constructs in these transformants was verified by Southern blot analysis and positive transformants were crossed with the fluG strain RBN119 (WIESER and ADAMS 1995 ) to generate fluG; alcA(p)::fluG strains.

Supplemented minimal media for growth of A. nidulans was prepared as described (KAFER 1977 ). Complete medium is minimal medium supplemented with yeast extract at 5.0 g/liter. Threonine shift time course experiments with alcA(p):: fluG strains were carried out as described (LEE and ADAMS 1995 ). The effect of different nitrogen sources on conidiation of dsgA1 mutant strains was determined by inoculating 5 x 10⁵ conidia/ml into 100 ml of minimal medium containing different sources of nitrogen equivalent to the amount present at 2.0 g/liter of yeast extract. The nitrogen sources tested were glutamine (6.3 g/liter), ammonium tartarate (8.0 g/liter), bactopeptone (Difco; 7.8 g/liter), casein hydrolysate [Sigma (St. Louis); 9.2 g/liter], L-arginine (4.5 g/liter), L-asparagine (6.5 g/liter), and L-glutamic acid (14.6 g/liter). Cultures were examined microscopically for conidiation following 16 hr of growth at 37°. The degree of conidiation observed in the fluG; dsgA1 strain varied, but was always either diminished or reduced relative to the dsgA1 strain under the same conditions, indicating some level of inhibition. Sterigmatocystin (ST) production (shown in Table 3) and stcU mRNA levels (Fig 3) in strains grown in liquid complete medium was determined from 1- to 4-day-old stationary cultures as described earlier (HICKS et al. 1997 ).

The dsgA1 mutant was isolated by 4-nitroquinoline-1-oxide (NQO) mutagenesis of the homozygous fluG strain DCD1 using a protocol described earlier (WIESER et al. 1994 ). Spores were mutagenized to a survival rate of 42% and mutants were screened on complete medium with 0.004% Triton-X-100. The fluffy phenotype of a fluG strain is not rescued on complete medium as on minimal medium, facilitating a visual screen for conidiating mutants. Strains fluG; dsgA1 (HMDCD1.4), fluG⁺; dsgA1 (HDCD3.8), and flbA; dsgA1 (HDCD4.26) were obtained by haploidization of the diploids MDCD1, DCD3, and DCD4, respectively, by treatment with the microtubule destabilizing agent benomyl (HASTIE 1970 ). Presence of fluG or flbA alleles was checked by Southern analysis. The fadA^Q204L; dsgA1 strain was constructed by transforming an argB; dsgA1 strain (HDCD9.1) with plasmid pJYPK26 (YU et al. 1999 ) bearing the fadA^Q204L allele and argB as a selectable marker. To assess growth rates of dsgA1 mutants, the radii of three different colonies of each strain were determined from the center of the colony to two different margins (n = 6) over a period of 5 days of growth on minimal medium. We report the mean values for radial growth rates and standard deviation calculated at 95% confidence levels.

Mitotic mapping of dsgA1 was carried out by constructing diploids between a fluG; dsgA1 mutant and various strains with scorable markers on different chromosomes as shown in Table 2. Cosegregation of the fluG phenotype with the wild-type allele of a given mapping marker served as an indicator of the absence of dsgA1 on the corresponding chromosome, thereby facilitating mapping of dsgA1 by a process of elimination. Haploid segregants of diploid strains were isolated following treatment with benomyl and assortment of the dsgA1 mutation with mapping markers was followed. Phenotypes scored include FlbA (fluffy autolytic), W (white spores), FluG (fluffy aconidial), Met (methionine requirement), Lys (Lysine requirement), S (inorganic sulfate requirement), His (histidine requirement) and Fwa (fawn spores). From data in Table 2, it can be seen that among haploid segregants of benomyl-treated diploids DCD13 and DCD14, dsgA1 does not show free recombination with the sB3 and lysA1 markers on chromosome VI, indicating that dsgA1 is located on chromosome VI.

Nucleic acid isolation and manipulation:
Samples collected for RNA analysis were frozen in liquid nitrogen, lyophilized, pulverized, and extracted with TRIzol reagent as specified by the manufacturer (GIBCO BRL, Gaithersburg, MD). Fifteen micrograms of RNA/lane were separated by electrophoresis on formaldehyde-agarose gels, transferred directly onto nylon membrane (Hybond-N; Amersham, Arlington Heights, IL), and hybridized to ³²P-labeled random probes. A 2.5-kbp XhoI fragment from the plasmid pFM1 (ADAMS et al. 1992 ) was used as a fluG-specific probe.

Oligonucleotides used in this study were engineered to include restriction enzyme sites (underlined) and are listed below:

• G1: 5'-CAG AAT GGG GAT CCT ACC ATT GA-3'

• BN9: 5'-AGG AGA AAG CTT AGA CTC-3'

• BN16: 5'-GAG AGA GTG GGG ATC CCG ATG AAC CAG-3'

• BN17: 5'-GTC GAG CTC GAC GCT G-3'

• OL1: 5'-CCA GAC GGA TCC CCG TAT CTC GTC-3'

• OL2: 5'-CAA TAC CTC TCG AGA AGC CAC TTC CTG-3'.

To overproduce the C-terminal GSI FluG domain a 1.5-kbp fluG(387–865) fragment, synthesized by PCR using oligonucleotides G1 and BN9, was inserted into a BamHI-HindIII- digested pBN55 vector (LEE and ADAMS 1995 ). This resulted in placement of fluG(387–865) under alcA promoter control in the plasmids pBN67 and pBN68. Since the alcA::fluG(387–865) construct complemented fluG and resulted in the overproduction of FluG protein of the expected size (detected by Western blot) we did not sequence the PCR-generated fluG(387–865) allele.

To construct the vector for production and purification of FluG(387–865) from Escherichia coli, a 1.4-kbp region corresponding to the FluG-coding sequence was PCR amplified using a cDNA clone as a template and oligonucleotides OL1 and OL2 as PCR primers. The resulting product was digested with BamHI and XhoI and inserted into the plasmid pET-27b(+) (Novagen) to create pCD2. To delete the N-terminal region of fluG and place the C-terminal region under the control of its own promoter, plasmid pCD9 was constructed as follows: A 1.1-kbp SacI-BamHI fragment, bearing sequence upstream of the FluG open reading frame, was PCR amplified using oligonucleotides BN16 and BN17. This fragment was inserted into pBN67 (see above) upstream of 1.5-kbp of the C-terminal fluG region, to produce pCD9. Plasmid pCD18 bearing the full-length fluG gene under the control of its own promoter was constructed by first inserting the 2.6-kbp SacI-HindIII fragment of pCD9 into pET-27b(+) (Novagen) from which a similar sized XbaI-XhoI fragment was inserted into pPK1 (provided by Pat Kennedy and Dr. Lawrence Yager, Temple University, Philadelphia), a vector bearing the argB gene. The resulting construct was called pCD13. An internal 1.0-kbp PstI fragment of pCD13 encompassing the N-terminal deletion was replaced by a 2.3-kbp PstI fragment from the genomic fluG clone pFM1 (ADAMS et al. 1992 ) to produce pCD18.

Microscopy and thin layer chromatography (TLC) analysis:
Photomicrographs of hyphal development were taken using an Olympus BH2 compound microscope and differential interference contrast optics. ST was extracted from stationary cultures and subjected to TLC chromatography and detection as described earlier (HICKS et al. 1997 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The N terminus of FluG is dispensable for conidiophore development:
Our earlier work demonstrated that fluG was required for wild-type sporulation and that fluG overexpression could cause conidiation in submerged culture, a condition that normally represses development. Several fluG mutations that produce a fluffy aconidial phenotype were found to reside in the C-terminal GSI-like domain (amino acids 387–865) of FluG (LEE and ADAMS 1994A ) and therefore we tested the hypothesis that this region would be sufficient to direct development. Strain RCD47.18 was constructed lacking the wild-type fluG gene but bearing a portion of the fluG gene encoding the C-terminal GSI domain under the control of the inducible alcA promoter. The aim was to test whether FluG(387–865) could function like full-length FluG in causing conidiophore development when overproduced in submerged vegetative hyphae. Overexpression of fluG(387–865) caused submerged conidiation similar to that observed following overexpression of the full-length fluG gene (Fig 1A). Reduced conidiophores with spores at the ends of hyphal tips were seen as early as 9 hr postinduction (not shown), and fully developed conidiophores bearing vesicles and sterigmata were present by 24 hr after alcA induction. Western blot analysis using anti-FluG(387–865) antibody verified that a 53-kD C-terminal polypeptide was indeed overproduced in the fluG; alcA::fluG (387–865) strain.

Gene overexpression can produce gain-of-function mutations that provide insight into function. However, it is important to recognize that overexpression mutations may result in phenotypes that have little to do with protein function under normal conditions. To address this issue we tested whether the C-terminal half of FluG was also sufficient to cause conidiation when expressed from the native fluG promoter under growth conditions that support wild-type conidiation. To this end, we constructed the strain fluG + fluG(387–865) bearing a copy of the fluG gene from which codons 1–386 were specifically deleted but containing its own upstream regulatory sequences, integrated at the argB locus. As shown in Fig 1B, the fluG + fluG(387–865) strain conidiated similarly as a fluG strain complemented with a wild-type fluG(1–865) allele, indicating that the N-terminal region of FluG is not required for normal conidiophore development.

dsgA1 dominantly suppresses the conidiation defect of a fluG mutant:
To better understand how FluG functions in activating conidiation, we isolated fluG suppressor mutations to identify genes that act downstream of fluG. Given that fluG mutations could be partially suppressed by nutritionally limiting growth conditions, we expected that many types of recessive, growth-retarding mutations could result in suppression due to complex nutritional effects. To avoid this, we set out to isolate dominant fluG suppressor mutants that would identify activators of the FluG signaling pathway. Colonies of NQO mutagenized, diploid homozygous fluG mutant cells (DCD1) were screened for the ability of cells to conidiate on complete medium. One strong suppressor mutant (MDCD1) and 67 relatively weaker suppressors were found among 116,000 surviving colonies. The weak suppressors were set aside for later study while the strong suppressor mutation was designated as dsgA1 and characterization of this locus is described below.

Treatment of the diploid dsgA1 mutant with the microtubule destabilizing agent benomyl (HASTIE 1970 ) resulted in formation of both fluffy and conidial haploid segregants, as expected if the suppressor was dominant. To further test dominance we constructed a diploid between a conidial haploid fluG; dsgA1 segregant (HMDCD1.4; Fig 2A, left) and a fluG mutant strain. The resulting diploid strain was conidial, thereby confirming dominance of dsgA1 (not shown). All strains were examined by Southern blot analysis to confirm the absence of a wild-type fluG gene (data not shown). dsgA1 was mapped to chromosome VI as described in MATERIALS AND METHODS.

Close examination of the fluG; dsgA1 strains indicated numerous differences from the fluG⁺ wild-type strain. The fluG; dsgA1 strains displayed a hyperconidial phenotype in which conidiophores not only arose from specialized aerial mycelium but also formed on the basal mycelium on the surface of the agar (not shown). Through observation of the dsgA1 mutant and wild-type colonies originating from single spores and growing on an agar surface, we found that dsgA1 mutants produced recognizable conidiophores within 12 hr of placing a spore on media while wild-type strains required 22 hr to reach a similar developmental point. This rapid conidiophore development by dsgA1 mutants was not limited to colonies growing on agar surfaces but also occurred with similar timing when mutants were grown in liquid shake culture (Fig 2B).

Additionally, we found that fluG; dsgA1 mutants had other defects relative to a fluG⁺ strain. The fluG; dsgA1 mutant (HMDCD1.4) exhibited reduced radial growth rate (4.41 + 0.33 mM/day) in comparison to the fluG⁺ strain (TTA11; 6.64 + 0.196 mM/day) and the fluG strain (TTA127.4; 6.35 + 0.34 mM/day). The dsgA1 strains also displayed a dominant defect in sexual development. Although fluG; dsgA1 mutants readily formed heterokaryons with developmentally wild-type strains, these heterokaryons did not produce cleistothecia under growth conditions that support wild-type sexual reproduction. This reproductive defect is conditional, however, in that dsgA1 strains can cross with wild-type strains when grown as heterokaryons on minimal plates with 20 mM glycine as the sole source of nitrogen and 2% glucose as the carbon source.

dsgA1 causes hyperconidiation independently of fluG:
To examine interactions between the dsgA1 mutation and fluG, a fluG⁺; dsgA1 mutant strain was constructed (HDCD3.8; Fig 2A, left). The fluG⁺; dsgA1 strain also displayed reduced radial growth rates (3.95 + 0.09 mM/day) relative to the fluG⁺ strain (TTA11; 6.64 + 0.196 mM/day) and had a hyperconidial phenotype that was even more pronounced than that seen for fluG; dsgA1 mutants. We concluded that under these conditions the dsgA1 phenotype prevails independently of fluG function, supporting the hypothesis that dsgA1 is a gain-of-function mutation in a gene that has a positive influence on conidiation.

Observation of submerged conidiation by dsgA1 mutants allowed us to detect an interesting difference between fluG; dsgA1 mutants and fluG⁺; dsgA1 strains. While both strains sporulated between 9 and 11 hr after inoculation in defined minimal medium (Fig 2B), only the fluG⁺; dsgA1 strain sporulated during growth in minimal medium supplemented with yeast extract (Fig 2B), bactopeptone, or other high quality nitrogen sources such as glutamine or ammonium (not shown). This inhibitory effect was not observed when strains were grown exposed to air.

dsgA1 suppresses flbA and fadA activating mutations:
One role we have proposed for FluG is to inactivate FadA, a G-subunit of a heterotrimeric G-protein that stimulates vegetative growth (YU et al. 1996 ) by inducing the RGS activity of FlbA. Both loss-of-function flbA mutations and dominant activating fadA mutations cause a fluffy autolytic phenotype due to constitutive and uncoordinated induction of hyphal growth (LEE and ADAMS 1994B ; YU et al. 1996 ). We wanted to determine if dsgA1 bypasses the need to inactivate FadA-mediated signaling (i.e., Do dsgA1 mutants activate sporulation even when FadA is activated constitutively?). To address this question, flbA; dsgA1 and fadA^Q204L; dsgA1 double mutant strains were constructed. Both flbA; dsgA1 (HDCD4.26) and fadA^Q204L; dsgA1 (TJYPK26.15) were now able to conidiate, indicating that the dsgA1 mutation is able to overcome the requirement for inactivation of FadA in stimulating conidiophore development (Fig 2A, right). However, in liquid minimal medium conidiation was observed in the fadA^Q204L; dsgA1 strain but not in the flbA; dsgA1 strain (Fig 2B), possibly because FlbA has a FadA-independent activity in stimulating conidiation under these conditions (YU et al. 1996 ; HICKS et al. 1997 ).

fluG and flbA sterigmatocystin biosynthesis defects are not suppressed by dsgA1:
Several Aspergillus species produce the carcinogenic polyketides, aflatoxin and ST, that are detrimental to both health and the economy. The genes required for synthesis of the aflatoxin precursor ST in A. nidulans have been found to reside within a 60-kbp gene cluster (stc; BROWN et al. 1996 ). Recent data have revealed that fluG and flbA genes are required for ST production, and dominant activating mutations in fadA disrupt ST biosynthesis (HICKS et al. 1997 ). It has been hypothesized that ST production is dependent on the inactivation of FadA by FlbA, which in turn is stimulated by FluG activity. Given the ability of dsgA1 to suppress the aconidial phenotypes conferred by fluG, flbA, and dominant activating fadA mutations, we were interested in determining the effect of dsgA1 on ST production in these mutant backgrounds. As indicated in Table 3, dsgA1 strains produce ST in a fluG- and flbA-dependent manner. Thus, while the dsgA1 mutation suppresses defects in conidiation of the fluG and flbA null mutants, the dsgA1 mutation does not suppress their requirements for ST production. Additionally, it does not alleviate the inhibition imposed by fadA dominant activating mutations on the ST production pathway. Examination of the expression of stcU (one of the genes in the stc cluster) by Northern analysis (Fig 3) further indicated that the dsgA1 mutation does not bypass the need for fluG in stc gene expression. These results support the conclusion that dsgA1 is exclusively involved in the asexual conidiation pathway.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The A. nidulans fluG gene plays a pivotal role in the switch from vegetative growth to initiation of conidiation and is hypothesized to cause this by inducing the production of an extracellular factor(s) (ADAMS et al. 1992 ; WIESER et al. 1994 ). While FluG is a bipartite protein, conidiation-inducing activity is confined to the GSI-like C-terminal half of the protein. We have shown that overexpression of the C-terminal half of FluG is sufficient to cause development in submerged culture. Moreover, expression of a C-terminal derivative fluG(387–865) under the control of its own promoter was sufficient to complement a fluG mutant. These results demonstrate that the C-terminal region of FluG has a distinct function in development even at wild-type levels of expression.

Although the critical part of FluG is related to glutamine synthetase I, FluG appears to have no role in glutamine biosynthesis because fluG mutants are not glutamine auxotrophs (LEE and ADAMS 1994A ) and an endogenous glutamine synthetase exists in A. nidulans (MACDONALD 1982 ; CORNWELL and MACDONALD 1984 ). We propose that the GSI region of FluG has a unique role in extracellular factor biosynthesis, perhaps catalyzing an enzymatic reaction very similar to that carried out by glutamine synthetases (GS). Alternatively, FluG may promote conidiation by degrading a conidiation inhibitory factor. In support of this hypothesis, recent BLAST search (ALTSCHUL et al. 1990 ) results revealed significant matches of the C-terminal FluG region to a Pseudomonas putida amino-group transfer protein TdnQ (e value = 1e^-12; FUKUMORI and SAINT 1997 ) and a GS-like component of the aniline dioxygenase complex from Acinetobacter sp (e value = 3e^-12; FUJII et al. 1997 ; TAKEO et al. 1998 ), both of which are involved in degradation of aromatic amines like aniline to catechol. From these comparisons, it also seems possible that FluG may catalyze a reaction(s) similar to an amino-group transfer.

Although the N terminus of FluG is not required for development, two protein sequences were found in the database that have significant similarity to this region of FluG. These include nodulin 6 from the plant Medicago truncatula (28% identity, 49% similarity), associated with the infection of its roots with Sinorhizobium meliloti (MATHIS et al. 1999 ), and a nodulin-like protein of unknown function from Arabidopsis thaliana (29% identity, 47% similarity). It is noteworthy that similarity with the Arabidopsis protein extends to the full-length FluG protein, suggesting the presence of a FluG homolog in plants. The significance of these similarities is not readily apparent but suggests that the N terminus of FluG may have some unknown function that is not essential for asexual development. Identification of the function of this A. thaliana protein might provide some useful insights into the function of FluG.

Regardless of the specific mechanism of FluG action, its function is required for both initiating conidial development and inhibiting mycelial growth. Initiation of asexual development in A. nidulans is dependent on the balance of two interacting pathways that respectively signal vegetative growth and conidiation (Fig 4). There is an increasing body of evidence to indicate that cross-talk between these pathways shifts the balance in favor of either growth or sporulation (ADAMS and TIMBERLAKE 1990 ; ADAMS et al. 1992 ; LEE and ADAMS 1994A , LEE and ADAMS 1995 ; WIESER and ADAMS 1995 ). Besides fluG, the asexual development pathway is also regulated by the early conidiation genes flbA, flbB, flbC, flbD, and flbE that are all required for the expression of brlA, encoding the primary activator of conidiation-specific genes. On the other hand, the vegetative growth pathway is in part regulated by the heterotrimeric G-protein -subunit FadA along with the Gß- subunit (ROSEN et al. 1999 ). FluG is hypothesized to inhibit vegetative growth by signaling activation of FlbA, which in turn interferes with the FadA-mediated growth-signaling pathway to promote conidiation (YU et al. 1996 ). Dominant or recessive loss-of-function fadA mutations suppress the requirement of flbA but not fluG in conidiation, indicating that FluG has a FadA-independent, development-specific role in addition to its proposed role in downregulation of growth. Initiation of development may therefore depend on both functions of FluG.

To gain further insight into the role of FluG in development, we also performed a genetic screen for dominant suppressors of fluG with the aim of isolating development-specific mutations that would help identify components acting downstream of FluG in the asexual development pathway. We identified dsgA1 as a dominant suppressor of the fluG fluffy aconidial phenotype. Observations of dsgA1 strains grown in submerged cultures indicate that it not only bypasses the need for FluG but also the requirement for air, the only known extrinsic factor that induces the genetically programmed, FluG-dependent pathway. A plausible mechanism of fluG suppression by dsgA1 would be the constitutive activation of a conidiation pathway component acting downstream of FluG, possibly a receptor of the extracellular factor or a signal transduction component such as a conidiation pathway specific G-protein. Alternately, the dsgA1 mutation could identify a component of a third, as yet uncharacterized, pathway that activates sporulation in a FluG-independent manner.

In addition to suppressing the fluG deletion mutation, we were surprised to learn that the dsgA1 mutation was able to suppress the loss-of-conidiation phenotypes observed in dominant activating fadA and flbA deletion mutant strains. This result raises the possibility that DsgA functions as part of the FadA growth-signaling pathway rather than having a direct role in sporulation. However, other mutations that are known to inactivate FadA signaling, such as fadA (YU et al. 1996 ) and sfaD (ROSEN et al. 1999 ) that otherwise suppress flbA mutants, are unable to suppress fluG mutants. Therefore, we propose that DsgA functions directly in activating sporulation and that the dsgA1 mutation stimulates such a strong activation of the sporulation pathway that conidiation takes place while FadA-mediated growth signaling remains active. In keeping with this hypothesis, we found that dsgA1-directed sporulation is enhanced by the presence of the wild-type fluG gene, possibly reflecting a DsgA-independent role for FluG in promoting sporulation by inhibiting FadA-mediated growth signaling. This requirement for FluG may also be reflected in the observation that conidiation of a dsgA1 mutant in liquid complete medium and in media containing rich nitrogen sources is fluG dependent.

In addition to the mycelial proliferation pathway, the FadA G-protein also regulates biosynthesis of ST, the penultimate precursor of the fungal metabolite aflatoxin, in that FadA inhibits stc gene expression and ST production (YU et al. 1996 ; HICKS et al. 1997 ; ADAMS et al. 1998 ). The early developmental regulators FluG and FlbA are also required for ST production by negatively influencing FadA activity in a similar way as in asexual sporulation. FluG is believed to promote the production of this secondary metabolite via inactivation of FadA signaling. Although the dsgA1 mutation bypasses the need for fluG and flbA in conidiation, and circumvents the inhibition of the dominant activating fadA^Q204L allele on this process, dsgA1 fails to restore ST biosynthesis in these mutant strains. Thus, the dsgA1 mutation specifically influences asexual development, and as indicated in the model shown in Fig 4, characterization of dsgA1 mutants has further clarified the function of interacting signaling pathways in regulating conidiation under different environmental conditions in A. nidulans and the influence of FluG on this regulation. How DsgA fits into this model awaits the isolation and characterization of the dsgA gene.

	FOOTNOTES

¹ Present address: Department of Genetics, Duke University Medical Center, Durham, NC 27710.
² Present address: Syngenta Agricultural Discovery Institute, Inc., San Diego, CA 92121-1125.
³ Present address: Monsanto, Mystic, CT 06355.

	ACKNOWLEDGMENTS

We thank Jenny Wieser, Stefan Rosén, and Dr. Joseph Heitman for comments on this manuscript and our colleagues in the laboratory for advice and helpful discussions. This work was supported by a National Institutes of Health grant GM-45252 to T.H.A.

Manuscript received October 3, 2000; Accepted for publication April 18, 2001.

	LITERATURE CITED

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