toxins has significant health and economic costs. We A.nidulans ST pathway has led to identification of a 60 kb gene cluster (the ST Cluster; stc) that includes 25 coThe filamentous fungus Aspergillus nidulans contains regulated genes, many of which have been shown to a cluster of 25 genes that encode enzymes required to function in ST biosynthesis (Brown et al., 1996). Transynthesize a toxic and carcinogenic secondary metabolscription of all of these genes is dependent upon the ite called sterigmatocystin (ST), a precursor of the activity of aflR, a pathway-specific regulatory gene found better known fungal toxin aflatoxin (AF). One ST within the ST cluster that encodes a zinc binuclear clusterCluster (stc) gene, aflR, functions as a pathway-specific type DNA binding protein (Woloshuk et al., 1994; Yu transcriptional regulator for activation of other genes et al., 1996a). aflR expression is regulated during the life in the ST pathway. However, the mechanisms concycle such that aflR mRNA begins to accumulate early in trolling activation of aflR and synthesis of ST and AF the stationary phase and activation of other genes required are not understood. Here we show that one important for ST biosynthesis quickly follows (Yu et al., 1996a). level for control of stc gene expression requires genesNumerous observations have supported the hypothesis that were first identified as early acting regulators of that microbial secondary metabolite production and sporulasexual sporulation. Specifically, we found that lossation are intimately associated. The best known example of-function mutations in flbA, which encodes a RGS is found in Streptomyces spp., in which there are common domain protein, or dominant activating mutations in elements that regulate sporulation and antibiotic producfadA, which encodes the α subunit of a heterotrimeric tion (for a review see Hopwood, 1988). Similarly, earlier G protein, block both ST production and asexual observations suggested that the ability to complete wildsporulation. Moreover, overexpression of flbA or type asexual sporulation could be a prerequisite for ST/AF dominant interfering fadA mutations cause precocious biosynthesis in the filamentous fungus genus Aspergillus stc gene expression and ST accumulation, as well as (Kale et al., 1994(Kale et al., , 1996. To study the genetic basis unscheduled sporulation. The requirement for flbA in of the relationship between asexual development and sporulation and ST production could be suppressed by secondary metabolism and the possible common elements loss-of-function fadA mutations. The ability of flbA to in regulation of these two processes, we decided to activate stc gene expression was dependent upon examine the effects of various early acting developmental another early acting developmental regulator, fluG, mutations on ST production in A.nidulans. We previously and AflR, the stc gene-specific transcription factor.proposed that there are two antagonistic signaling pathThese results are consistent with a model in which ways regulating A....

The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the G␣ subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl cyclase. Taken together, these studies further define the cAMP signaling cascade controlling virulence of this ubiquitous human fungal pathogen.Cryptococcus neoformans is a heterothallic, basidiomycetous, pathogenic fungus that causes serious infections of the central nervous system in individuals immunocompromised by AIDS or undergoing organ transplantation, cytotoxic chemotherapy, or corticosteroid therapy (6, 31). The virulence of C. neoformans is influenced by several factors, including the production of an antiphagocytic polysaccharide capsule (7,20,27), the use of melanin as an antioxidant (26, 51), growth at host physiological temperature (37 to 39°C) (27, 29), and prototrophy (41). Although not directly involved in the virulence of C. neoformans, mating and filamentous growth may play a survival role in the environment and also promote dissemination of the pathogen into the host. Signaling cascades regulating virulence and differentiation of C. neoformans have been extensively studied, including a mitogen-activated protein kinase (MAPK) pathway, a G-protein-regulated cyclic AMP (cAMP) pathway, a Ras-specific pathway, and the calcineurin pathway (for reviews, see references 29 and 48).The MAPK cascade regulates mating processes involving morphological differentiation, such as the dikaryotic mycelia, basidia, and basidiospores, which are produced in response to peptide pheromones secreted by opposite mating-type cells (24,32,43). The MAPK pathway is composed of mating-typespecific (Ste3␣/a, Ste20␣/a, Ste11␣/a, and Ste12␣/a) and nonspecific (Gpb1, Ste7, and Cpk1) elements (12, 49). Gene disruption experiments revealed that the MAPK pathway is required for mating and cell type-specific differentiation but not for virulence (12). However, mating type has been associated with the virulence of serotype D (variety neoformans) strains by Kwon-Chung et al., who showed that the ␣-mating type is more virulent than the a-mating type (25). Furthermore, Del Poeta et al. demonstrated that the MF␣1 pheromone gene is induced during the late stages of central nervous system infection...

, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.

Filamentous fungi elaborate a complex array of secondary metabolites, including antibiotics and mycotoxins. As many of these compounds pose significant economic and health concerns, elucidation of the underlying cellular mechanisms that control their production is essential. Previous work revealed that synthesis of the carcinogenic mycotoxins sterigmatocystin (ST) and aflatoxin (AF) in Aspergillus species is negatively controlled by FadA, the α‐subunit of a heterotrimeric G‐protein. In sharp contrast, we show here that the dominant activating fadA allele, fadAG42R, stimulates transcription of a gene from the A. nidulans penicillin (PN) gene cluster and elevates penicillin production. Thus, FadA has opposite roles in regulating the biosynthesis of a potent antibiotic (PN) and a lethal mycotoxin (ST) in A. nidulans. Furthermore, expression of fadAG42R in Fusarium sporotrichioides increases trichothecene (TR) mycotoxin production and alters TR gene expression. Our findings reveal that a G‐protein defines an important control point for differential expression of fungal secondary metabolites within and across fungal genera. These data provide critical evidence suggesting that targeting G‐protein signal transduction pathways as a means of controlling or preventing the production of a single mycotoxin could have serious undesirable consequences with regard to the production of other secondary metabolites.

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 (ΔrcoA), 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 ΔrcoA strain. Overexpression of aflR in a ΔrcoA 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 ΔrcoA 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.