The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development.

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that is required for control of mycelial proliferation and activation of asexual sporulation. We identified a dominant mutation in a second gene, fadA, that resulted in a very similar phenotype to flbA loss-of-function mutants. Analysis of fadA showed that it encodes the alpha-subunit of a heterotrimeric G protein, and the dominant phenotype resulted from conversion of glycine 42 to arginine (fadA(G42R)). This mutation is predicted to result in a loss of intrinsic GTPase activity leading to constitutive signaling, indicating that activation of this pathway leads to proliferation and blocks sporulation. By contrast, a fadA deletion and a fadA dominant-interfering mutation (fadA(G203R)) resulted in reduced growth without impairing sporulation. In fact, the fadA(G203R) mutant was a hyperactive asexual sporulator and produced elaborate sporulation structures, called conidiophores, under environmental conditions that blocked wild-type sporulation. Both the fadA(G203R) and the fadA deletion mutations suppressed the flbA mutant phenotype as predicted if the primary role of FlbA in sporulation is in blocking activation of FadA signaling. Because overexpression of flbA could not suppress the fadA(G42R) mutant phenotype, we propose that FlbA's role in modulating the FadA proliferation signal is dependent upon the intrinsic GTPase activity of wild-type FadA.     

Extragenic suppressors of loss-of-function mutations in the aspergillus FlbA regulator of G-protein signaling domain protein.

We showed previously that two genes, fl bA and fadA, have a major role in determining the balance between growth, sporulation, and mycotoxin (sterigmatocystin; ST) production by the filamentous fungus Aspergillus nidulans. fadA encodes the alpha subunit for a heterotrimeric G-protein, and continuous activation of FadA blocks sporulation and ST production while stimulating growth. fl bA encodes an A. nidulans regulator of G-protein signaling (RGS) domain protein that antagonizes FadA-mediated signaling to allow development. To better understand FlbA function and other aspects of FadA-mediated growth control, we have isolated and characterized mutations in four previously undefined genes designated as sfaA, sfaC, sfaD, and sfaE (suppressors of flbA), and a new allele of fadA (fadAR205H), all of which suppress a fl bA loss-of-function mutation ( fl bA98). These suppressors overcome fl bA losses of function in both sporulation and ST biosynthesis. fadAR205H, sfaC67, sfaD82, and sfaE83 mutations are dominant to wild type whereas sfaA1 is semidominant. sfaA1 also differs from other suppressor mutations in that it cannot suppress a fl bA deletion mutation (and is therefore allele specific) whereas all the dominant suppressors can bypass complete loss of fl bA. Only sfaE83 suppressed dominant activating mutations in fadA, indicating that sfaE may have a unique role in fadA- fl bA interactions. Finally, none of these suppressor mutations bypassed fl uG loss-of-function mutations in development-specific activation.     

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

flbA encodes an Aspergillus nidulans RGS (regulator of G protein signaling) domain protein that antagonizes FadA (G(i)alpha-subunit of heterotrimeric G protein)-mediated growth signaling to allow asexual development. We previously defined and characterized five suppressors of flbA (sfa) loss-of-function mutations and showed that one suppressor (sfaB) resulted from a novel dominant-negative allele of fadA. In this report we show that a second suppressor gene (sfaD) is predicted to encode the beta subunit of a heterotrimeric G protein. Deletion of sfaD suppressed all defects resulting from complete loss-of-flbA function mutations, caused a hyperactive sporulation phenotype and severely reduced vegetative growth. However, the sfaD deletion could not suppress the growth activation caused by dominant-activating fadA alleles, indicating that constitutively active FadA can cause proliferative growth in the absence of Gbetagamma signaling. We propose that SfaD and FadA are both positive growth regulators with partially overlapping functions and that FlbA has an important role in controlling the activities of both proteins. Inactivation of signaling events stimulated by both components of the heterotrimeric G protein is essential for both sexual and asexual sporulation.     

RcoA has pleiotropic effects on Aspergillus nidulans cellular development

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain. Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR. CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.     

Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway

The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (ΔstcJ), an early ST intermediate, norsoloroinic acid (ΔstcE) or a late ST intermediate, versicolorin A (ΔstcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.     

Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans.

We showed previously that a DeltafluG 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 ( approximately 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 DeltafluG(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 DeltaflbA 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.     

FluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlA beta activation.

The Aspergillus nidulans fluG gene is necessary for the synthesis of a small diffusible factor that is required for the endogenously regulated induction of asexual sporulation that takes place during the development of an air-exposed colony. Previous work established that FluG is present at nearly constant levels throughout the Aspergillus life cycle, leading to the hypothesis that FluG factor is constitutively produced and development initiates after its concentration surpasses a fixed threshold. Here we show that overexpression of fluG can overcome the developmental block normally imposed on vegetative cells in submerged culture and leads to the formation of complex conidiophores that are remarkably similar to wild-tye conidiophores made by air- exposed colonies. This fluG-induced sporulation requires the activities of other early developmental regulatory genes including, flA, flB, flC, flD, flE, and brlA. The requirement for flbA in fluG-induced sporulation is particularly interesting because overexpression of flbA can also induce sporulation in submerged culture and this flbA activity requires fluG. The interdependence of fluG and flbA activities suggests a close relationship between the products of these two genes in controlling conidiophore development. In addition to the endogenous sporulation signal provided by fluG, several environmental factors, including air exposure, carbon or nitrogen stress, and increased osmolarity, can influence developmental activation. We demonstrate that each of these signals requires the brlA beta gene, but not brlA alpha, to initiate conidiophore development. We present a model to account for the complex genetic and environmental controls leading to the activation of brlA beta and sporulation.     

The push-pull mechanism of bacteriophage Ø29 DNA injection

Regulators of G-protein signalling play a crucial role in controlling the degree of heterotrimeric G-protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (DeltargsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, DeltargsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G-protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by DeltargsA were suppressed by deletion of ganB encoding a Galpha subunit, indicating that the primary role of RgsA is to control negatively GanB-mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS-Galpha pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.