INTRODUCTION

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Asexual sporulation is a common reproductive mode for a diverse group of fungi that includes many medically, industrially, and agriculturally important species. Asexual spores of higher fungi are called conidia, and although there is great variety in conidial form and function, all conidia represent nonmotile asexual propagules that are usually made from the side or tip of specialized sporogenous cells and do not form through progressive cleavage of the cytoplasm. The process of conidiation involves many common developmental themes including temporal and spatial regulation of gene expression, cell specialization, and intercellular communication. The genetic mechanisms controlling these processes have been addressed in any detail for only two well-studied ascomycetes, Aspergillus nidulans and Neurospora crassa. Although the general mechanisms governing sporulation in these two fungi are similar, there are clearly important differences. This review will concentrate on the progress made over the last few years toward understanding the genetic regulation of development in A. nidulans. A recent review by Springer (144) describes the N. crassa conidiation pathway.

From a fairly simplistic point of view, the A. nidulans asexual reproductive cycle can be divided into at least three different stages, beginning with a growth phase that is required for cells to acquire the ability to respond to induction signals, proceeding through initiation of the developmental pathway, and culminating with execution of the developmentally regulated events leading to sporulation. It is possible to at least partially separate these stages in the laboratory because the vegetative or hyphal state is continuously maintained during submerged growth in nutritionally sufficient liquid medium (14). After cells have acquired the ability to respond to inducing signals, development can be induced synchronously by exposing hyphae to air (see below). The kinetics of conidiation under these conditions are highly reproducible (14, 84), and this approach can be used for obtaining large quantities of developmentally staged materials for biochemical analysis.

There are numerous other experimental features that have made A. nidulans a particularly useful organism with which to study sporulation and development in general (153). Conidiation can easily be observed macroscopically due to conidial pigmentation; because conidiophores and conidia are dispensable structures, it is simple to obtain mutants that are defective in their ability to produce normal conidiophores. The fact that A. nidulans can reproduce sexually as well as asexually means that, once generated, most conidiation-deficient mutants can be manipulated to construct strains with desired genotypes for epistasis studies or for mapping mutations to specific locations on one of the eight chromosomes (42, 74, 126). A. nidulans also has a well-defined parasexual cycle providing additional tools for genetic analysis (74, 126). Hundreds of genes have been mapped to specific locations by classical methods, and it is typically quite simple to map a newly identified mutation to a given chromosome by using parasexual genetics and then to determine its linkage to previously defined genes by meiotic analyses (39, 155). With the advent of electrophoretic karyotyping, it is also possible to map cloned genes to determine their chromosomal locations by probing blots of resolved chromosomes (22). In addition, a physical map of overlapping cosmids from the two commonly used cosmid libraries has been constructed and provides even simpler tools for gene mapping (23) (available at http://fungus.genetics.uga.edu:5080/Physical_Maps.html).

As with yeast and many other fungi, a significant advantage of genetic studies of A. nidulans comes from the ready availability of sophisticated molecular genetic tools for specific functional analysis. DNA-mediated transformation with genomic DNA libraries allows relatively straightforward isolation of genes through complementation of mutant phenotypes followed by plasmid or cosmid recovery (154). Because transformation often results in homologous integration, it is feasible to disrupt, delete, or otherwise modify specific genes in the genome (1, 104, 150). Transcriptional promoters can be analyzed in vivo by fusing the promoter of interest to a reporter gene like the Escherichia coli lacZ gene and introducing the construct into the fungus (158). -Galactosidase activity can be determined at various times during development by isolating crude protein from cell extracts. In situ staining is also possible and is one way to determine the cellular location of a given protein (5, 7). Recently, several laboratories have shown that the Aequorea victoria gene encoding green fluorescent protein (GFP) (128) can be used in A. nidulans to examine protein expression and localization in living cells. However, to date this approach has been successful only when GFP is expressed at very high levels with strong promoters. Several strong inducible promoters that can drive the expression of a gene of interest are available and are useful for studying the developmental effects of misscheduled gene expression (1, 63, 90).

MORPHOGENESIS AND SPORULATION

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Colony Formation

Vegetative growth in A. nidulans, as in other filamentous fungi, begins with the germination of a spore. This could be either a mitotically derived conidiospore or a meiotically produced ascospore. Spore germination leads to the formation of tubular hyphae that grow in a polar fashion by apical extension and branching to form a network of interconnected cells known as a mycelium. The mycelium forms a radially symmetric colony that expands indefinitely at a constant rate of about 0.5 mm h¹ at 37°C (87). Macroscopically, the fungal mycelium appears to be an amorphous collection of equivalent vegetative cells. Functionally, however, the various cells within the mycelium interact to form an ordered network with different hyphae or cells presumably playing distinct roles in the acquisition of nutrients from the environment and in determining the precisely timed development of specialized reproductive structures. While various parameters affecting mycelial development have been described for numerous fungi (129), the molecular mechanisms controlling hyphal growth and colony formation remain largely uncharacterized. Substantial research aimed toward understanding basic cell biological problems associated with hyphal growth, including cell cycle, cytokinesis, and polarity determinants, has been performed, but this is beyond the scope of this review (15, 102, 122, 167).

Approximately 16 h after spore germination, the first phenotypic evidence of hyphal specialization within the colony becomes readily apparent (35, 87). At this time, aerial hyphal branches are formed in the center of the colony and some of these branches subsequently differentiate into conidiophores, the term applied to the asexual spore bearing structures of A. nidulans. It takes between 6 and 8 h from the initiation of aerial growth to the formation of the first asexual spore or conidium, so that a new spore is formed and the asexual cycle can be reinitiated within about 24 h after the original spore germinates (35). Following formation of the first conidiophores within the center of the colony, developmental initiation moves out toward the edge of the colony, leaving the oldest conidiophores in the center and the newly forming conidiophores at the margin of the growing colony. While it takes approximately 16 h of growth for hyphae in the center of the colony to become competent to develop the first conidiophores, newly forming hyphal elements in the radially expanding colony are able to elaborate conidiophores very soon after their appearance. Given that the radial expansion rate of an A. nidulans colony growing on complete medium at 37°C is about 0.5 mm/h and conidiophores can be observed within 1 to 2 mm of the colony margin, it can be surmised that these structures are formed within 2 to 4 h of the appearance of the new hyphae. This is particularly interesting in that the cell cycle in A. nidulans is about 100 min (112) and conidiophores contain dozens of nuclei. Presumably, such rapid formation of conidiophores requires recruitment of nuclei from hyphae, but it could also involve uncharacterized alterations in the cell cycle. Although A. nidulans will also grow vegetatively in liquid submerged culture, with few exceptions (see below) such a culture is aconidial. Typically, hyphae must be exposed to an air interface to stimulate conidiophore production (33, 84).

Termination of asexual reproduction begins in the center of the colony and is followed by commencement of another clearly recognizable developmental process, meiotic (or sexual) spore formation. This stage involves the formation of multicellular fruiting bodies, called cleistothecia, that are surrounded by specialized cells termed Hülle cells (35). Maturation of the sexual fruiting body includes karyogamy and meiosis within the specialized ascogenous hyphae found inside the cleistothecium and concludes with formation of hundreds of asci, each containing eight binucleate ascospores (35, 169). The mechanisms controlling cleistothecial development have not been studied as thoroughly as for conidiation. However, there are many indications that important interactions occur between the asexual and sexual sporulation pathways in A. nidulans (described below). As with conidia, ascospore germination results in the regeneration of the vegetative hyphae and reinitiation of the lifecycle.

The formation of conidiophores in a colony is a complex process that can be divided into several morphologically distinct stages (Fig. 1) (153). This process begins with the growth of a conidiophore stalk that elongates by apical extension of an aerial branch, much like vegetative hyphal growth (Fig. 1A). The conidiophore stalk differs from a vegetative hyphae in at least three easily recognizable ways (109, 153). First, the stalk cell extends from a specialized thick-walled cell, termed a footcell, that anchors the stalk to the growth substratum. Because it is difficult to identify a footcell in the mycelium before a stalk is observed, it is not really known whether the formation of the thick-walled footcell actually precedes the formation of the stalk, as is typically stated. In maturing conidiophores, the footcell can be distinguished from other mycelial cells by the presence of a two-layered wall in which the outer layer is continuous with the rest of the mycelium while the inner layer is unique to the footcell and the emerging conidiophore stalk (109). The second distinguishing feature of the conidiophore stalk is that it has a 4- to 5-µm diameter as opposed to the 2- to 3-µm diameter more typical of vegetative aerial hyphae. Finally, unlike vegetative aerial hyphae that grow indefinitely and are capable of branching, conidiophore stalks rarely branch and their length is relatively determinate. For A. nidulans, the conidiophore stalk attains a height of about 100 µm, but this is a species-specific trait and varies among the different species of aspergilli.

View larger version (76K): [in this window] [in a new window]   FIG. 1.   Morphological changes during conidiophore formation. Shown are scanning electron micrographs of the stages of conidiation. (A) Early conidiophore stalk. (B) Vesicle formation from the tip of the stalk. (C) Developing metulae. (D) Developing phialides. (E) Mature conidiophores bearing chains of conidia. Reproduced from reference 109 with permission of the publisher.

After apical extension of the conidiophore stalk ceases, the tip begins to swell and reaches a diameter of ~10 µm (Fig. 1B). This structure, known as the conidiophore vesicle, is not separated by a septum from the stalk, so that the stalk, vesicle, and footcell essentially comprise a single unit. Mims et al. (109) observed multiple nuclei aligned around the outside of the vesicle that undergo a more or less synchronous division associated with the synchronous production of buds from the vesicle surface to form a layer of primary sterigmata termed metulae (109). However, Fischer and Timberlake (58) reported that the nuclei underwent several synchronized mitoses while the conidiophore stalk was elongating and the vesicle was expanding whereas nuclear movement into the buds was a more gradual process. Microscopic analysis of conidiophore heads indicates that each contains about 60 metulae and that each metular bud forms a cigar-shaped cell about 6 µm long and 2 µm wide that contains a single nucleus (40, 109, 119) (Fig. 1C). Metulae in turn bud twice to produce a layer of about 120 uninucleate sterigmata termed phialides. This metular budding is polar so that both phialides are produced from the end of the metula that is farthest from the vesicle (Fig. 1D). The production of primary and secondary sterigmata in this way has been compared to pseudohyphal growth of Saccharomyces cerevisiae, and some similarities in the genes involved in these processes have been observed (62; see below). Although phenotypically like metulae in size and shape, phialides differ from metulae in that they give rise to chains of uninucleate spores called conidia (Fig. 1E). In this respect, phialides have been likened to stem cells because they undergo repeated asymmetric division to produce chains of spores, but maintain their own identity (153). Phialides also play a supportive role during conidiation producing products that function only in the spore (100, 118, 146, 153). Because each phialide can make upward of 100 spores, the total number of conidia from one conidiophore probably exceeds 10,000. Like stalk height, the formation of sterigmata differs among Aspergillus species (48). Some species lack metula and produce phialides directly from vesicles while others produce more than one layer of metula before making phialides. How and why this diversity in conidiophore structure among closely related fungi developed is an interesting evolutionary problem but has not been addressed experimentally.

PHYSIOLOGICAL REQUIREMENTS FOR CONIDIATION

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Acquisition of Developmental Competence

Conidiation does not usually occur in A. nidulans until cells have gone through a defined period of vegetative growth (14, 33). This was demonstrated by taking advantage of the fact that under normal media conditions, A. nidulans can be maintained indefinitely in the vegetative stage of its life cycle by growing hyphae submerged in liquid medium. Asexual sporulation only rarely takes place and sexual sporulation never occurs unless vegetative hyphae are exposed to air. This trait provides a means for synchronous developmental induction by harvesting hyphae grown in submerged culture and then exposing them to air (13, 84). By varying the times for which hyphal cultures are maintained in submerged culture, it has been shown that there is a minimum growth period before cells can respond to induction (14, 170). If development is induced following at least 18 h of vegetative growth, the time required to observe the initial conidiophore-specific structures remains constant at about 5 h (Fig. 2). If the submerged growth period is shorter than 18 h, the time required for development increases correspondingly. These results have been interpreted to mean that cells require approximately 18 h of growth before they are competent to respond to the inductive signal provided by exposure to air.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Experimental determination of the timing of developmental competence (Tc). Spores were inoculated into liquid medium at time zero and allowed to germinate in submerged culture. At the times indicated on the horizontal axis, mycelia were transferred to a solid substrate and observed to determine the earliest time of conidiophore formation. The delay time (d) is the interval between transfer to solid medium and the first appearance of conidiophores. The time Tc is the time after which d = D = constant. Reproduced from reference 35 with permission of the publisher.

Although the acquisition of competence is the earliest described conidiation-specific event in the A. nidulans life cycle, it is also the least understood. Neither the concentration of a limiting nutrient such as glucose nor continuous transfer to fresh medium alters the timing with which cells become competent (33, 123). However, precocious conidiation mutants with a decreased time requirement for the acquisition of competence (14, 51) have been isolated, suggesting that there is a genetic component to competence. Physiological changes ranging from differences in the inducibility of enzyme systems to altered glucose uptake have been correlated with cultures obtaining developmental competence, but how these changes are regulated and which events occur first are not known (33, 123). In this regard, it is interesting that these physiological changes take place more quickly in precocious conidiation mutants than in wild-type strains. Taken together, these results have been taken to support the hypothesis that this early aspect of Aspergillus conidiophore development occurs as an integral part of the life cycle rather than as a response to unfavorable environmental conditions.

As mentioned above, developmentally competent liquid-grown hyphae from A. nidulans typically do not conidiate until after they are exposed to air (14). The mechanism by which exposure of competent hyphae to air serves to induce conidiation is not understood. The signal is probably not associated with changes in O₂ or CO₂ levels but has been proposed to involve cell surface changes induced by abrupt formation of an air/water interface at the hyphal surface (113). It is important to note that the conidiation block in submerged culture is not absolute and is dependent on the strain as well as the medium composition (96, 135, 141). Experiments aimed at understanding the genetic events leading to the initiation of conidiation are described later in this review.

Conidiation in A. nidulans strains that have the wild-type allele of the velvet gene (veA⁺) is red light dependent in that veA⁺ A. nidulans strains do not conidiate as effectively in the dark as veA1 mutant strains do, even if exposed to air (111). However, 15 to 30 min of continuous exposure to red light at any time after induction and before the formation of differentiated structures apparently activates the developmental program. Exposure to far-red light immediately after exposure to red light at least partially suppresses the activation of conidiation. This photoreversibility of red light induction is reminiscent of phytochrome-mediated responses observed in higher plants, leading to the suggestion that a phytochrome-like molecule in A. nidulans may regulate development. A. nidulans strains carrying the veA1 mutation conidiate in the presence or absence of light, suggesting that the veA product may play a negative regulatory role in the absence of light. Thus, VeA may function similarly to numerous plant genes that are required to prevent photomorphogenesis in dark-grown plants and in roots (111).

While sporulation by air-exposed A. nidulans colonies apparently occurs in a medium-independent fashion, there have been many reports describing A. nidulans conidiation in submerged culture under conditions where nutrients are limited or in response to other stresses (96, 113, 135). Most recently, Skromne et al. (141) showed that A. nidulans will sporulate following transfer from nutritionally complete medium to medium lacking either a nitrogen or a carbon source for growth (Fig. 3). Although these conditions are apparently sufficient to cause development in submerged culture, it is not yet clear whether such nutritional stress plays a direct role in developmental activation for hyphae grown on the surface of a plate. In fact, nutrient limitation reduces the overall conidiation of surface colonies and poor supplementation of many auxotrophic mutants makes them nearly asporogenous (136; see below). Moreover, continual replacement of the growth medium beneath a surface-grown colony does not inhibit sporulation (123). Thus, while nutritional stress may be sufficient to cause sporulation, it is apparently not required for development to take place. These observations might be explained if the development of conidiophores in wild-type colonies exposed to air occurs in response to an internal signal that activates the sporulation pathway in a genetically programmed way (see below). This signal either is absent or fails to function in submerged culture. However, in the absence of this signal, other environmental cues such as carbon and nitrogen starvation can cause developmental induction. In any case, it is interesting that as with development on the surface of a plate, development of submerged cultures induced by metabolic stress apparently requires that cells go through a period of vegetative growth to become competent before transfer to nitrogen or carbon-free medium will cause development (86, 141).

View larger version (102K): [in this window] [in a new window]   FIG. 3.   Starvation can induce conidiation in submerged culture. A wild-type strain was grown in glucose minimal medium for 18 h and then shifted to fresh minimal medium (A) or minimal medium lacking a carbon source (B). Conidiophores formed within 12 h after a shift to nitrogen starvation medium (development also occurred upon shifting to medium lacking a nitrogen source), but no conidiophores were seen in the culture shifted to minimal medium containing a usable carbon or nitrogen source. Reproduced from reference 86 with permission of the publisher.

Differentiation of the multiple cell types making an A. nidulans conidiophore involves the controlled activation of several hundred genes (151-153). However, to date the functions of only a small number of these genes has been determined. Timberlake showed that there are approximately 1,200 diverse mRNAs that accumulate to varying concentrations specifically during conidiation (151). By contrast, Martinelli and Clutterbuck used mutational analysis to estimate that only 45 to 100 genes are uniquely required for asexual sporulation (98). This large discrepancy in predicted gene number could be explained if some genes detected based only on expression patterns encode redundant or incremental functions that would not be detectable by simple visual examination of mutants. In keeping with this idea, deletion from A. nidulans of a 38-kbp region containing numerous spore-specific genes did not result in detectable changes in development (10). Furthermore, subtle defects in sporulation, like the spore wall defect resulting from deletion of the rodA gene (see below), might be difficult to detect in broad screens based on tedious visual examination of colonies (146). Finally, Martinelli and Clutterbuck (98) limited their analysis to mutations that did not alter vegetative growth rates. mRNAs present at high levels during sporulation may also be present at low levels in vegetative cells and may be required for some aspect of normal growth and metabolism. In fact, many cDNAs isolated as conidiation-specific genes have turned out to contain genes involved in carbon metabolism or other cellular processes (115). Mutations in these genes would have been excluded by Martinelli and Clutterbuck. Regardless of the actual number of loci involved, it is certain that many genes are specifically required for sporulation and that, at least in some cases, their transcription is developmentally controlled.

Genetic and biochemical studies of A. nidulans asexual sporulation have led to the identification of only two genes, brlA and abaA, that are specifically required for conidiation (without affecting vegetative growth) and are absolutely necessary for spore formation (45). Together with a third gene called wetA, brlA and abaA have been proposed to define a central regulatory pathway that acts in concert with other genes to control conidiation-specific gene expression and determine the order of gene activation during conidiophore development and spore maturation (21, 110). Mutations in any one of these three genes blocks asexual sporulation at a specific stage in conidiophore morphogenesis and prevents expression of a broad class of developmentally regulated mRNAs (21, 110).

brlA encodes an early regulator of development. The phenotype of brlA null mutants has been described as "bristle" because the early developmental block in these mutants prevents the transition from polar growth of the conidiophore stalk to swelling of the conidiophore vesicle (45). Instead, brlA mutants differentiate conidiophore stalks that grow somewhat indeterminately, reaching heights 20 to 30 times taller than wild-type conidiophores and giving the colony a "bristly" appearance (Fig. 4). These brlA mutants also fail to accumulate developmentally regulated transcripts including abaA and wetA mRNAs (21). Thus, brlA is proposed to become necessary for activating development-specific gene expression beginning at the time of conidiophore vesicle formation. Two lines of evidence indicate that brlA activity continues to be required up through and including spore formation. First, numerous hypomorphic mutant alleles of brlA that support more extensive conidiophore development than brlA null mutants, differentiating complex structures with different degrees of vesicle and sterigmata development but never spores, have been described (42, 47). These hypomorphic mutants presumably maintain partial BrlA function and result in altered expression patterns for numerous genes encoding development-specific activities. Second, developmental induction experiments with a temperature-sensitive brlA allele, brlA42^ts, showed that development was arrested following a shift to the restrictive temperature for BrlA42 activity, regardless of the developmental stage (110).

View larger version (135K): [in this window] [in a new window]   FIG. 4.   brlA mutants form indeterminate conidiophore stalks. Conidiophores from a wild-type strain (A) and a brlA mutant strain (B) are shown. In the wild-type strain, the stalks grow to a fairly uniform height and bear additional conidiophore-specific structures, while the stalks of a brlA mutant grow somewhat indeterminately and fail to elaborate other conidiophore-specific cells. Arrows indicate stalks. Reproduced from reference 1 with permission of the publisher.

View larger version (87K): [in this window] [in a new window]   FIG. 5.   Overexpression of brlA activates sporulation in submerged culture. The brlA gene was placed under the control of the alcohol-inducible promoter alcA. The alcA(p)::brlA strain and a wild-type strain were grown for 12 h in liquid minimal medium containing glucose to repress brlA expression from the alcA promoter and then shifted to alcA(p)-inducing medium. (B) By 3 h after the medium shift, the alcA(p)::brlA strain produced spores from the tips of hyphae. (A) No conidiation was observed in the wild-type strain even 24 h after the medium shift.

Activation of abaA initiates a positive-feedback loop. abaA encodes a developmental regulator that is activated by brlA during the middle stages of conidiophore development after sterigmata differentiation (9). The phenotype of abaA null mutants is described as "abacus" because these mutants produce aconidial conidiophores that differentiate sterigmata but do not form sporogenous phialides (45, 137) (Fig. 6). Instead, these mutants form branching sterigmata, leading to long chains of cells that appear like beads on a string as in an abacus. Although the brlA transcript accumulates to relatively normal levels in abaA mutants, many other developmentally regulated mRNAs (including the wetA mRNA) do not.

View larger version (79K): [in this window] [in a new window]   FIG. 6.   Conidiophores from a wild-type strain (A) and an abaA mutant strain (B). The abaA mutant produces normal conidiophore stalks (ST) and vesicles (VS), but metulae (M) and phialides are abnormal and form abacus (AB) structures instead of conidia (C). Magnifications for panels A and B are equivalent. Reproduced from reference 110 with permission of the publisher.

wetA is required late in development. The wetA gene is required late in development for the synthesis of crucial cell wall components (95). The phenotype of a wetA null mutant is described as "wet-white." These mutants differentiate normal conidiophores, but the conidia produced are defective in that they never become pigmented but instead autolyse (138).

stuA and medA modify development. Two other developmental regulatory genes, stuA and medA, have been shown to be necessary for the precise spatial pattern seen in the multicellular conidiophore (6, 28, 107, 108). stuA and medA have been termed developmental modifiers and are required for a restricted series of cell divisions that establish the spatial organization of the conidiophore. Mutations in either gene give rise to spatially deranged conidiophores, but both stuA and medA mutants are able to produce some viable conidia and are therefore termed oligosporogenous mutants (45).

View larger version (114K): [in this window] [in a new window]   FIG. 7.   Electron micrographs of stuA and medA mutants. In the stuA mutant, abnormal conidia are formed either directly from the conidiophore vesicle (A) or from abnormal sterigmata (B and C). Normal metula and phialides are not produced. medA mutant conidiophores are initially nearly normal, but multiple layers of metulae are produced before phialides differentiate and begin to form conidia (D and E). Sometimes sterigmata redifferentiate to produce secondary conidiophores (F). Reproduced from reference 6 with permission of the publisher.

(i) Gene classes. By examining patterns of RNA accumulation in a series of brlA, abaA, and wetA mutant strains during normally induced development or following alcA(p)::brlA, alcA(p)::abaA, or alcA(p)::wetA induction, the nonregulatory developmentally activated genes were divided into four categories (95, 110, 153). Class A genes are activated by either brlA or abaA or both, independent of wetA (Fig. 8). These class A genes are expected to be involved in early development events. abaA alone activates wetA, which in turn activates Class B genes, independent of brlA and abaA. Thus, class B genes are expected to encode spore-specific functions. Class C and D genes require the combined activities of brlA, abaA, and wetA for their expression and have been proposed to encode phialide-specific functions. Class C and D genes are distinguished from one another by their expression patterns during normally induced development in wild-type and mutant strains. Accumulation of wetA mRNA requires wetA⁺ activity both during normal conidiophore development and in forced-expression experiments, bringing up the interesting possibility that wetA is autogenously regulated (21, 95).

View larger version (12K): [in this window] [in a new window]   FIG. 8.   Timeline of conidiation and the central regulatory pathway. The genes in the central regulatory pathway for conidiation are predicted to activate other genes responsible for the production of conidiophores. The activation of the class A, B, C, and D genes give rise to the formation of a conidiophore with the timing indicated in the upper part of the figure.

(ii) Pigmentation genes. The characteristic dark green pigment of wild-type A. nidulans spores results from the production of a spore-specific pigment that gives colonies a green appearance and provides a straightforward means of identifying mutants with abnormal pigmentation (38, 41, 45, 126). These mutants have allowed the identification and characterization of genes involved in pigment synthesis that serve as excellent reporters of developmentally regulated gene expression (155). Although many loci contribute to the nuances of spore color (see below), two genes, wA and yA, are central to the process of spore pigmentation. The wA gene encodes a polyketide synthase, and mutations in wA result in white spored strains that lack melanin and an electron-dense outer spore wall layer that contains -1,3-glucan (99, 100). yA is predicted to encode a p-diphenol oxidase known as conidial laccase, and yA mutants have yellow spores (12, 38, 84, 171). The fact that wA mutations are epistatic to mutations in yA and that conidial laccase accumulates in the walls of wA mutants has led to the hypothesis that wA catalyzes the synthesis of a yellow pigmented polyketide that is converted to the green form by the product of the yA gene (100). Although these products have not been characterized in A. nidulans, Brown et al. isolated and characterized a compound from a laccase-deficient strain of Aspergillus parasiticus and showed the compound to be an octaketide (24). The transcripts of both yA and wA are developmentally regulated by genes from the central regulatory pathway. wA is apparently regulated as a class B gene (above) in that it is directly controlled by the developmental regulatory gene wetA (99). Both brlA and abaA can direct the activation of yA, making this a class A gene (11, 110).

(iii) Spore wall protein genes. Another class of genes with auxiliary functions that are regulated during conidiophore development encodes proteins that make up specific components of the spore wall. Two A. nidulans genes, rodA and dewA, have been shown to encode hydrophobic proteins that contribute to the hydrophobicity of conidia, presumably facilitating their dispersal by air (146, 147). Both of these genes were isolated from a group of cDNA clones called CAN (conidiation A. nidulans) that were isolated based on their developmentally regulated expression (21, 174). RodA and DewA belong to a general class of proteins found in fungi, termed hydrophobins, that have been found in several species including members of the Basidiomycetes, Ascomycetes, and Deuteromycetes (161). Like dewA and rodA, other fungal hydrophobins were identified principally because they are abundantly produced during various cellular differentiation processes. The predicted proteins have little sequence identity at the amino acid level but have certain structural features in common. Most importantly, they all contain eight cysteine residues arranged in a conserved pattern and they have a similar arrangement of hydrophobic domains and thus exhibit similar hydrophobicity plots (54). Hydrophobin-encoding genes in other fungi function in a wide range of processes including spore (16, 83) and hyphal (114, 134, 160) wall hydrophobicity and infection structure formation in plant (148) and insect (145) pathogens.

(iv) Other sporulation-related gene functions. Many of the mutants isolated from early screens for conidiation-specific genes were not only defective in aspects of conidiophore morphogenesis but were also at least partially defective in their vegetative growth properties (42, 45, 98). This implies that some cellular or metabolic functions are more critical during conidiation than during vegetative growth. For example, Champe's group isolated a mutant that was unable to form asexual spores on complete medium (medium containing the chemically undefined supplement yeast extract), but this conidiation defect could be remediated by addition of arginine to the medium (136). On defined minimal medium (medium without yeast extract), arginine was required for growth but at a much lower concentration than that required for conidiation. Genetic analysis of this mutant showed it to be an allele of the argB locus, which encodes the enzyme ornithine transcarbamylase. Thus, what initially appeared to be a conidiation-specific defect turned out to be a defect in a metabolic process whose function is in greater demand during conidiation (136). In their survey of conidiation mutants, Martinelli and Clutterbuck (98) also isolated auxotrophic mutants for which partial starvation has a more deleterious effect on vegetative growth. These mutants may identify metabolic processes that are critical for asexual sporulation but do not identify genes that are specific to development.

View larger version (162K): [in this window] [in a new window]   FIG. 9.   apsA mutants produce anucleate sterigmata. Electron micrographs showing conidiophore formation in a wild-type strain (A to D) are contrasted with micrographs of an apsA mutant (E to H). In apsA mutants, development of the conidiophore is normal up to the metula stage (E and F). Nuclei typically fail to enter the metulae, and development arrests with an anucleate metular bud (F). Occasionally, nuclei enter metulae, leading to production of functional phialides and normal chains of spores (G and H). Reproduced from reference 58 with permission of the publisher.

As described above, Prade and Timberlake (127) demonstrated that mutations separately eliminating brlA or brlA activity caused developmental abnormalities but that additional doses of brlA in a brlA mutant strain or of brlA in a brlA mutant strain remediated these defects. These results have been interpreted to mean that the two brlA-encoded polypeptides have redundant functions and led to the hypothesis that brlA and brlA have evolved to provide separate means of responding to the multiple regulatory inputs controlling brlA expression (68, 127). Han et al. (68) constructed translational fusions between each of the brlA and brlA ORFs and the E. coli lacZ gene to begin to test this hypothesis. They found that brlA and brlA are regulated in different ways (Fig. 10). The brlA mRNA is transcribed in vegetative cells before developmental induction but does not accumulate to appreciable levels, presumably because translation of the brlA µORF represses BrlA translation to block development. The importance of this translational block was demonstrated by eliminating the µORF AUG, which led to brlA expression in hyphae and inappropriate sporulation. This result implies that one way in which brlA expression could be initiated following induction is through a controlled change in the choice of translational initiation sites from the µORF AUG to the BrlA AUG. However, it is also possible that increased transcription of brlA provides sufficient template to allow inefficient BrlA translation from the internal AUG. In support of the latter hypothesis, it has been demonstrated that forced activation of brlA transcription in hyphae is sufficient to cause sporulation despite the presence of the µORF. In addition, there is a significant increase in brlA mRNA levels following developmental induction. Although this has not been shown to result from increased transcription, specific fragments from the brlA regulatory region have been shown to confer developmentally specific expression to an otherwise inactive promoter-lacZ fusion (69). In any case, the ultimate result of brlA activation is activation of other developmentally specific genes. Following BrlA translation, brlA transcription is activated primarily through the brlA-dependent positive feedback loop by a mechanism that probably involves AbaA and may also involve BrlA directly. This prediction is supported by the observation that 5 AbaA and 12 BrlA binding sites are found upstream of brlA (8, 36). We also found that forced overexpression of brlA resulted in the accumulation of brlA mRNA whereas forced activation of brlA did not result in brlA accumulation (68, 69).

View larger version (10K): [in this window] [in a new window]   FIG. 10.   Model for differential control of brlA and brlA during conidiophore development. The brlA mRNA is transcribed in vegetative cells before developmental induction, but translation of the µORF represses translation of BrlA. Following induction, unknown regulatory factors activate BrlA translation from brlA by removing the translational block imposed by the µORF, increasing the transcription of brlA, or both. Activation of BrlA translation leads to transcription of abaA and other downstream regulatory proteins. This in turn activates a positive-feedback loop that leads to high levels of brlA expression to cause further developmental changes. Reproduced from reference 68 with permission of the publisher.

Fluffy mutations define early developmental regulators. In estimating the number of genes required for various stages in development, Martinelli and Clutterbuck proposed that 83% of all sporulation mutants were altered in their ability to initiate development before formation of the conidiophore vesicle and presumably before activation of brlA expression (98). This result indicates that regulation of the decision to initiate conidiophore development is highly complex, requiring the activities of numerous genes (98). The most common phenotype observed among these proposed early conidiation mutants has been described as fluffy. The term "fluffy" includes a diverse group of phenotypes, but in general all fluffy mutants grow as undifferentiated masses of vegetative hyphae forming large cotton-like colonies (56, 98, 149, 165). To begin to understand the role that fluffy genes have in controlling brlA activation, Wieser et al. undertook an extensive genetic analysis of both dominant and recessive mutations that cause a fluffy phenotype (165). The analysis of recessive fluffy mutations led to the characterization of six genes, designated fluG, flbA, flbB, flbC, flbD, and flbE. Loss-of-function mutations in any one of these six genes result in a fluffy colony morphology and greatly reduced brlA expression (Fig. 11). All of these genes have been physically isolated and studied in some detail (3, 85, 87, 162-164). Dominant mutations causing a fluffy phenotype have also been characterized with the thought that this approach could identify some important regulatory genes not found among the recessive fluffy mutants (166). Although these dominant mutants have proven difficult to study by classical genetic approaches, analysis of the mutants resulting from dominant activating mutations in fadA has proven useful for understanding the relationship between growth control and sporulation (173; see below).

View larger version (48K): [in this window] [in a new window]   FIG. 11.   Phenotypic classes of fluffy mutants. Colonies were grown for 3 days on solid minimal medium. (A) Developmentally wild-type strain of A. nidulans. (B) fluG mutant. (C) flbA mutant. (D) Delayed conidiation mutant typical of flbB, flbC, flbD, and flbE mutants.

(i) fluG is required for production of an extracellular factor and is related to prokaryotic glutamine synthetase I. Although fluG mutants produce fluffy colonies (3, 85, 165) (Fig. 11B) and fail to activate brlA under normal growth conditions, there are two ways in which they can be induced to conidiate. First, when fluG mutants (even a fluG deletion mutant) are grown on a solid surface under suboptimal nutritional conditions they produce some conidiophores (3). This result has been interpreted to indicate that fluG is required for a genetically programmed aspect of conidiophore development. In the absence of fluG, it is possible to detect sporulation in response to environmental stress. This environmentally regulated response presumably also occurs in the wild type but is overwhelmed by the more prolific fluG-dependent programmed development response. As predicted in this model, fluG mutants can respond to starvation stress in submerged culture by producing conidiophores (86). However, the response differs from that observed for the wild type in two respects. First, although elimination of the carbon source causes fluG mutants to sporulate in submerged culture, the number of conidiophores observed is smaller than in the wild type. Second, although elimination of the nitrogen source does cause morphological changes in the fluG mutant in submerged culture, it does not cause sporulation. Taken together, these results might indicate that there are two aspects to stress-induced sporulation in submerged cultureone that is fluG dependent and a second that is fluG independent. Because overexpression of fluG in submerged culture can cause development (see below), one aspect of stress-induced development could be increased activity of fluG.

View larger version (47K): [in this window] [in a new window]   FIG. 12.   Overexpression of fluG, flbA, and flbD causes conidiophore production in submerged culture. The alcA promoter was used to drive the expression of fluG, flbA, and flbD. The alcA(p)::fluG, alcA(p)::flbA, and alcA(p)::flbD strains and a wild-type strain were grown for 14 h in liquid minimal medium containing glucose to repress alcA and then shifted to alcA-inducing medium. Overexpression of fluG (B), flbA (C), and flbD (D) led to production of conidiophores in ~18 h, 9 h, and 9 h after the shift, respectively. The wild-type strain (A) never produced conidiophores or spores. Panel D is reproduced from reference 162 with permission of the publisher.

(ii) flbB, flbC, and flbD encode nucleic acid binding proteins. Another group of fluffy mutants can be distinguished by the observation that beginning 2 to 3 days after inoculation, conidiophores were produced in the center of the colony while the margins remained fluffy (Fig. 11D). All of the delayed-conidiation fluffy mutants characterized to date define four distinct complementation groups, designated flbB, flbC, flbD, and flbE (165). The wild-type genes were each isolated by complementation of the respective mutants (162-164). As described for fluG, the cloned flbB, flbC, flbD, and flbE genes recognize RNAs that are present in vegetative cells and continue to be expressed at relatively constant levels during development. flbD is predicted to encode a 314-codon polypeptide that includes a region with significant identity to the DNA binding domain in the human proto-oncogene c-myb (91, 162). flbC is predicted to encode a 354-amino-acid polypeptide (164) that includes two CC/HH zinc finger domains typical of a large group of DNA binding proteins including A. nidulans BrlA (1, 106). flbB is predicted to encode a 389-amino-acid polypeptide (163) with similarity to b-Zip DNA binding proteins (159). Thus, flbB, flbC, and flbD are likely to function as DNA binding proteins and could control the transcriptional activation of other developmental regulators, like brlA, in response to sporulation signals. To date, no similarities between flbE and other proteins in the available databases have been observed (163). As with fluG, overexpression of either flbC or flbD in submerged hyphae results in the activation of brlA expression and sporulation (Fig. 12D) (analyses of flbB and flbE overexpression are in progress), further supporting the hypothesis that these proteins play a direct role in initiation of development.

(iii) flbA antagonizes a G-protein-mediated signaling pathway that inhibits sporulation. The final group (Fig. 11C) of fluffy mutants analyzed is distinguished by the fact that the center of the colony begins to disintegrate by 3 days after inoculation and the entire colony has autolysed so that only a few hyphal strands remain by 5 days postinoculation (87, 165). All of the recessive mutant strains that have been identified as having this fluffy autolytic phenotype have been shown to result from mutations in a single locus designated flbA. The flbA gene produces a 3-kb mRNA that, like fluG, flbB,