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Current Protein and Peptide Science, 2010, 11, 704-718

Adaptative and Developmental Responses to Stress in Aspergillus nidulans Oier Etxebeste2, Unai Ugalde2 and Eduardo A. Espeso1,* 1

Dept. of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu, 9, 28040, Madrid, Spain; 2Department of Applied Chemistry, Faculty of Chemistry, University of The Basque Country. Manuel de Lardizabal 3, 20018, San Sebastian, Spain Abstract: Development in the ascomycete A. nidulans is principally determined by environmental signals. Adaptability to oxidative stimuli can derive in changes of growth patterns and/or the activation of sexual or asexual reproductive cycles but this model fungus might also respond to high osmotic or salt concentrations, the redox state, the availability and quantity of carbon or nitrogen sources and the degree or quality of illumination. Since each cell within the colony follows a single morphogenetic program at a time, all these environmental cues might be sensed and integrated into a limited number of intracellular signals which, finally, would activate the required morphogenetic program and repress the others. This signaling mainly occurs through stress response pathways. The present review aims to summarize the available knowledge on how these pathways transduce environmental stimuli to mediate morphological changes in Aspergillus nidulans.

Keywords: Calcium signaling, development, halo-osmotolerance, light response, morphogenesis, nutrient starvation, polarized growth, oxidative stress. 1. INTRODUCTION Morphogenesis encompasses programmed changes in gene expression patterns leading to the development of specialized cell types and, in higher eukaryotes, to the formation and organization of organs. Such transformations are often determined by a combination of endogenous and/or environmental stimuli. In eukaryotes, developmental transitions are commonplace from simple unicellular yeast to more complex mammalian embryogenesis and tissue development [1,2]. During embryogenesis, cells are initially proliferative and pluripotent, but become gradually restricted in terms of the range of cell fates into which they can develop. In adults, tissue stem cells are normally quiescent becoming proliferative upon injury [3]. However, little is known on how stimuli induce the morphological transformations and how the fate of a pluripotent cell is determined. In particular, Reactive oxygen species (ROS) have important implications in these changes [4] but other stimuli have to be also considered [5]. The study of “more simplistic” model organisms is a way to approach these questions and extend the knowledge on basic eukaryotic developmental mechanisms. Among these organisms are fungi, which spawn relatively few cell types and have, in addition, an economic or clinical importance. Neurospora crassa and Aspergillus nidulans are two fungal models representing a good platform for the study of development. The latter has been used worldwide with state of the art techniques for more than 60 years. It is a non pathogenic fungus, except for inmuno-suppressed individuals, but is phylogenetically related to a clinically important species (A. fumigatus) as well as species with economical or industrial value as A. niger or A. oryzae. Finally, the developmental *Address correspondence to this author at the Dept. of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu, 9. 28040, Madrid, Spain; Tel: 0034 91 837 31 12; Fax: 0034 91 536 04 32; E-mail: [email protected] 1389-2037/10 $55.00+.00

program of A. nidulans has been the object of intense and updated study from genetic, molecular, physiological and biochemical approaches, with clearly defined environmental stimuli that induce developmental change. This review summarizes the available information on how biotic and abiotic stimuli, mainly halo-osmotic and oxidative stresses, are sensed and transduced to modulate the transition from vegetative growth to asexual and sexual development. We will focus on the proteins that act in the transduction pathways and mainly on those that could enable the integration of the wide array of stimuli into a limited number of intracellular signals that will determine the morphological fate of cells. With this aim, we will initially introduce the main life-cycle phases of A. nidulans. 2. GENES BEHIND ASPERGILLUS NIDULANS DEVELOPMENTAL PROGRAMS Three developmental programs are considered in A. nidulans. Vegetative growth by polarized extension of cells, hyphae, is the dominant growth mode of filamentous fungi [6] and is activated after the selection of the polarity axis during spore germination [7,8]. Once the required machinery has been assembled the germ tube emerges and hyphal extension occurs exclusively through deposition of new material at the tip Fig. (1A, 1B and 1C) [7,9]. After three nuclear divisions, the first septum is generated [10]. Septation proceeds after mitosis, when the cell exceeds a determined volume, and is followed by the formation of lateral branches that generate new polar extension points Fig. (1C). These morphological transformations are controlled at the molecular level by sophisticated machinery that enables penetration into solid substrates (see summary of genes cited in this section in Suppl. Table 1). Understanding this mechanism and its relation with other developmental processes in response to external stimuli is one of the major challenges of modern fungal cellular biology [11]. © 2010 Bentham Science Publishers Ltd.

Adaptative and Developmental Responses to Stress in Aspergillus nidulans

Current Protein and Peptide Science, 2010, Vol. 11, No. 8

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Fig. (1). A) Schematic illustration of the main three morphogenetic programs in Aspergillus nidulans. B) A colony growing on solid medium may be divided into four differentiated regions, according to the main developmental program. Youngest region locates at the periphery (dark yellow) and comprises mainly submerged vegetative hyphae. At the inset, scale bar = 40 m. Light yellow region indicates aerial hyphae in which conidiophore production has been induced. (S) indicates stalk. At the inset, scale bar = 15 m. The green zone designates the region in which conidiophores (Con) are matured. At the inset, scale bar = 400 m. In the oldest, inner region (in blue) cleistothecia (C) and Hülle cells (Hc) are mainly found. Scale bar= 100 m. C) Diagrammatic depiction of germination, branching and polar growth processes (based on [7]). On the right, a drawing representing the main elements of the mechanisms required for polar growth maintenance at the fungal cell tip (based on [57]). D) Cellular modules composing the conidiophore, their timely formation scale and the genetic pathway controlling their formation (actualized from [13]). The sign “?” indicates a hypothetical transcriptional relationship. Scale bar = 10 m. E) Representation of the cleistothecia formation process (based on [16]).

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Extensive polarized growth may halt when entering asexual or sexual developmental programs. Conidiophores are the asexual reproductive structures bearing thousands of conidia [12,13], asexual propagules characterized by low water content and arrested metabolism. These features permit dispersal and survival under adverse environmental conditions [14]. Conidiophore development involves the generation of specific cellular modules called: stalk, vesicle, metulae, phialides and conidia Fig. (1A, 1B and 1D). Asexual cycle in A. nidulans is normally followed in time by the production of sexual reproductive structures, which contain meiospores called ascospores [15,16]. This morphogenetic program involves the formation of a characteristic structure called crozier Fig. (1A, 1B and 1E) [16]. First, a penultimate diploid cell is formed within the crozier by the fusion of two nuclei or karyogamy. Then, the last and the third cells fuse while the penultimate diploid cell follows subsequent meiotic and mitotic processes to form a structure, the ascus, which contains eight inmature ascospores. Ripening of the ascus involves a second mitosis and the completion of the formation of eight binucleate, red-pigmented, mature ascospores. Further development of ascii precedes cleistothecium formation. The process is repeated forming new croziers and then new cleistothecia. These sexual fruiting bodies are normally surrounded by Hülle cells. The exact function for these highly specialized cells remains unknown, however it was suggested that they provide nutrients to cleistothecia [17]. Classical and molecular genetic studies have provided with the basis to understand these three developmental programs of which details are given below. 2.1. Vegetative Growth Different types of plasma membrane receptor bound GTPases control the progression of development. Som and Kolaparthi [18] described that the initiation of vegetative growth was dependent upon an initial high level of active GTP-bound RasA (Rat sarcoma-GTP) [19] followed by its gradual decrease. Interestingly, the range of GTP- to GDPbound forms of Ras marks developmental stages. The Ras GTPase activating protein (GAP), GapA, controls the GTPto GDP- balance in A. nidulans and its activity is required during germination, vegetative growth and also during reproduction (see below; [20]). The Rho type GTPase Cdc42 also plays a key role in the activation of polar growth. In yeast, it is recruited to the polar-extension site by the guanine nucleotide exchange factor (GEF) Cdc24, thus promoting the transition from the GDPbound Cdc42 to the active GTP-bound form [21]. Consequently an accumulation of the active Cdc42-GTP form is usually found at the cell tip [22,23]. This process is promoted by the assembly of a complex in which both the GEF factor and the Cdc42 effector p21-activated kinase (PAK) associate with the scaffold protein Bem1 [24-28]. This stabilizes Cdc24 at the polarization site and, then, the Cdc42-GTP signal is transferred to multiple effectors that activate the formation of the actin cytoskeleton (see below). Finally, the GAPs Rga1, Bem2 and Bem3 induce Cdc42-GTP hydrolysis to generate the inactive GDP bound state. These Rho GAPs have a major effect on inhibiting polarized growth [29-31].

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This machinery is highly conserved among filamentous fungi [32], but important differences arise. In dimorphic fungi and some other molds, i.e.: Candida albicans or Ashbya gossypii, mutations in Cdc42 homologues cause lethality. In A. nidulans, two Rho-type GTPases play a role in polarity establishment: RacA and ModA, the Cdc42 homologue, being the latter not essential but might play an important function in branching at sub-apical cell compartments [33,34]. The downstream transfer of the Cdc42-GTP signal provokes the assembly of the Polarisome, a multiproteic complex that regulates the formation of cytoskeletal elements as actin cables and plays a key role in the activation and maintenance of polar growth [35]. A key component of the yeast polarisome is the formin Bni1, which serves as nucleation site for actin complexes [36-38]. Most of the polarisome components appear to spatially and temporally regulate Bni1 activity [37]. A. nidulans SepA, the Bni1 homologue, is maintained at polarization sites by the fungal protein MesA [39]. The homologue of the yeast polarisome component Spa2, SpaA, has also been observed at septation sites [40], suggesting the existence of mechanistic differences between S. cerevisiae and filamentous fungi. The Arp2/3 complex controls the formation of branched actin filaments, which are assembled near the plasma membrane at the cell apex. Components of this complex in yeast are, among others, for example Las17/Bee1, Arp2, Arp3 or Myo3 [41]. Myosins are actin dependent molecular motors and in particular A. nidulans MyoA, the yeast Myo3 homologue, is required for polarity establishment, actin cytoskeleton organization and for vesicle transport through actin cables [42]. Myosins are required in processes that are essential for the maintenance of polar extension, as endo- and exocytosis [43,44]. The latter comprise vesicle anchoring and their fusion with the plasma membrane, and is controlled by a protein complex called the exocyst [45]. In yeast, the exocyst contains eight members and interacts with further factors regulating polar growth, as for example Cdc42. The components of the exocyst complex are highly conserved among filamentous fungi and the presence of mutations in any of its components affect polarity establishment and cell growth [46]. Exocytosis requires the transport of cell wall building materials to the hyphal tip. Microtubules (MT) are used as molecular “highways” through which membranes, organelles, vesicles, diverse RNA molecules and protein complexes are transported [11]. In A. nidulans MTs seem to be required but are not essential for correct polar extension [47]. These cylindrical proteinaceous structures are nucleated (assembled) from the MT-organizing centers and extend by the fast addition of / dimers at the growing (plus) ends [11]. The opposite (minus) ends are less active or inactive [48]. Sudden MT depolymerization or shrinkage also occurs at the plus end, leading to a complete disappearance of MTs or to a switch back to elongation [11]. Recently, two major MT subpopulations have been identified. Detyrosinated MTs would maintain the tubule structure in mitosis while tyrosinated MTs would form the mitotic spindle [49]. Kinesins and dynein are molecular motors which transport a variety of cargoes through MTs, but in opposite directions. Cytoplasmic dyneins move towards minus ends of


MTs and kinesins towards the plus ends. Both types of molecular motors are essential for the correct positioning of the apical “body” or Spitzenkörper [50-52]. This fungal specific organelle precedes and directs the apical growth [53]. Multiple micro- and macrovesicles are transported from distal regions through MTs and accumulate at the Spitzenkörper, which is supposed to act as a vesicle supply center. At the cell tip, actin filaments enable the transport of vesicles from the Spitzenkörper to their final destination: the plasma membrane at the apex. [54,55]. Most of the vesicles are originated by exocytosis at the highly polarized Golgi (see above), while others contain plasma-membrane components recycled by endocytosis and accumulated at sub-apical regions Fig. (1C), right panel; [56,57]. On the other hand, the Spitzenkörper also accumulates ribosomes, which would allow the in situ translation of mRNAs specifically transported to the cell apex [58-61]. 2.2. Asexual Reproduction Conidiophore development begins when the stalk emerges and elongates from foot-cells [12]. Stalks are “low” specialized cells, 1-2 m wider than vegetative hyphae, without branches and with negative geotropism. These cells extend reaching a length of 100 m before their apical extension ceases [62]. Then the tip swells and forms a globular structure with a diameter of 10 m, called the vesicle. The stalk and vesicle bear no septa, but large vacuoles formed within the stalk condition the position of nuclei and probably alter cytoplasmic transport [63]. The following developmental step is the emergence of a layer of approximately sixty primary uninucleated sterigmata or metulae on the vesicle surface. This is a process that resembles a multipolar germination process of vegetative hyphae-like structures. Consistently, metula formation requires of vegetative extension modulators as, for example: the RasGAP protein, GapA [20], the Bem1p homologue polarisome component, BemA [64], or the GTPase RacA [34]. The elongation of metulae is followed by the apical division of each one to form a new layer of 120 secondary sterigmata or phialides. Primary and secondary sterigmata are morphologically divergent since each metula buds apically to form two phialides, and they in turn, give rise to long chains of conidia in a basipetal manner [65]. Classically, conidiophore development has been defined as an irreversible process upon activation [12,13]; references therein). Recently however, Etxebeste et al. [66] showed that the program yielding phialides from metulae could be aborted by imposing conditions that favored vegetative growth, resulting in miss-scheduled apical extension. Thus, metulae were re-defined as pluripotent cells able to follow at least two alternative differentiation pathways. Under normal aerial, oxidant, conditions a purported checkpoint sensing this oxidative environment would control the transition from metula to phialide formation. The genesis of the different conidiophore modules by the modified polar growth as well as budding processes is controlled at the genetic level by the central developmental pathway of conidiation (Suppl. Table 2). The C2H2-type transcription factor (TF) BrlA acts as the regulator [13] and the complexity of the corresponding brlA locus exemplifies


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the intricacy of this regulatory pathway. Two transcripts have been identified, brlA and brlA, both detected from the formation of the vesicle onwards [67]. brlA codes for the same peptide as brlA but with the addition of 23 amino acids at the N-terminal end. There is a third coding region, called ORF. The molecular role of each derived peptide and their functional relationship are unknown, but it has been described that brlA, brlA- or brlA- mutants are impaired at different stages of conidiophore development [13]. Moreover, it has been recently described that brlA expression is regulated by brlA [68]. These features suggest each brlA transcript exerts its function at different stages, being indispensable the correct spatial and temporal expression patterns to render a mature conidiophore bearing all cell types. AbaA is an ATTS type TF acting downstream of BrlA [69] and regulating phialide differentiation [65]. AbaA activates the expression of a wide array of genes, being wetA an important target. WetA is required at late stages of conidiophore development for the pigmentation and integrity of conidia [70]. Pigmentation of mature conidia with the characteristic green color requires the transcriptional activation of yA, encoding a p-diphenol oxidase or laccase [71]. Mutants in yA result in a distinctive yellow color. In addition, WetA participates in cell wall integrity by regulating the expression of class I and II chitin synthase genes, chsC and chsA [72]. brlA, abaA and wetA form the central developmental pathway of conidiation and their genetic products control the correct formation of conidiophores [73,74]. BrlA directs the expression of the others but during metula and phialide production AbaA also regulates its own expression [75]. Moreover, Aguirre [76] suggested that AbaA plays a negative feedback by direct repression of brlA expression at low levels, although at high levels it has a positive role. Nevertheless, WetA activity and the regulation of brlA expression are not linked [77]. The putative TF VosA controls trehalose biogenesis, spore maturation and has been recently characterized as a negative feedback regulator of brlA, thereby completing conidiation [78]. Other proteins have been proposed to modulate the spatio-temporal activity of the factors from the central conidiation regulatory pathway. Of these, indicate that StuA is an APSES type TF involved in their correct cellular distribution [79] and MedA is required for the temporal regulation of brlA expression [80,81]. 2.3. Sexual Reproduction The molecular mechanisms underlying the formation of sexual fruiting bodies are weakly understood [17]; see summary of genes in Suppl. Table 3), however, as a standardized technique in the laboratory, induction of sexual cycle involves the generation of oxygen and nutrient deprivation culture conditions. Regarding environmental sensing, six from the sixteen G-Protein Coupled Receptors (GPCR) identified in A. nidulans [82,83] have been characterized, and three of them, GprA, GprB and GprD, play a role in the control of sexual development [84,85]. GprD is a negative regulator of cleistothecium formation [84] while the GprA and GprB, acting downstream of GprD [85], are specifically required for self fertilization in homothallic conditions [83].


Receptor-bound G protein subunits are the first signal transduction elements in the control of sexual development [17]. Regulators of G protein signaling (RGS) catalyze the GDP-to-GTP exchange on the -subunit of heterotrimeric G proteins. In the GTP bound state occurs the dissociation of G from the and subunits, allowing G or G to activate downstream effectors independently [83]. In A. nidulans, G, G and G subunits FadA, SfaD and GpgA, and, specially, the RGS domain containing protein FlbA, play a key role in the appropriate control of development, activating downstream effectors of the required program but inhibiting the proteins of the other morphogenetic pathways [86-88]. Although they are not essential, the multiple effects on development found in deletion mutants suggest their central role in sexual differentiation. Cleistothecia production is determined by two distinctive signaling pathways, one mediated by cAMP (cyclic AMP)dependent protein kinases, and the other by mitogenactivated protein kinases (MAPK). Several studies described the sexual related phenotype of adenylate cyclase mutants in other fungi [89,90]. However, no perturbation of the A. nidulans sexual cycle has been reported in mutants lacking either the adenylate cyclase gene or the protein kinase A (PKA), despite the role in germination and hyphal growth displayed, respectively [91]. The sequential signal transduction by phosphorylation relying on the MAP kinase cascades is a widely described mechanism implicated in the regulation of gene expression in response to environmental stimuli. The High Osmolarity Glycerol (HOG; [92]) pathway is a major signaling pathway based on this transducing phosphomechanism. In A. nidulans, the HogA pathway is required for the induction of sexual development in addition to the response to reactive oxygen species and asexual osmoadaptative response. This pathway will be extensively covered in section 3.1. A number of diverse TFs are required for the negative or positive regulation of cleistothecia formation. The FadAdependent GATA-type NsdD and the zinc-finger type NsdC are key TFs that are independently required for cleistothecia or Hülle cell formation [86] by regulating cell wall construction at early stages of sexual development [93] or by repressing asexual development [94], respectively. In addition, cleisothecia and Hülle cell formation require the action of the leucine zipper-like protein encoded by dopA and the APSES type TF StuA [95]. An interconnection between asexual and sexual development is found with SteA, the homolog of the S. cerevisiae homeodomain-C2H2-zinc finger TF Ste12p, which represses medA expression. MedA functions as a temporal regulator of brlA expression in the asexual cycle (see previous section) and is required for cleistothecia but not for Hülle cell formation [96]. Finally, RosA and NosA are the two A. nidulans homologs of the Zn2 C6 TF Pro1 from the homothallic pyrenomycete Sordaria macrospora [97,98]. It has been proposed that RosA played a role in early decisions and the repression of sexual development while NosA activity would be required for primordium maturation. Both TFs are interconnected, as RosA seems to repress NosA expression. Sexual development also requires the participation of other general pathways. For example, Araújo-Bazán et al.

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[99] described the requirement of one of the main nuclear carriers, the importin- KapA, for the completion of sexual cycle. On the other hand, cytoskeletal elements such as one of the two -tubulin encoding genes, tubB, are essential for ascospore formation [100]. Overall, sexual reproduction requires the precise integration of diverse cellular processes [17] plus a highly sophisticated balance mechanism between both sexual and asexual reproduction cycles and vegetative mode. This demands the participation of proteins common to all pathways. 2.4. Cell Death, the Triple “A” Programmed cell death (PCD) processes have been associated with the main life-cycle phases of Aspergillus nidulans. This occurs, for example, in the case of autolysis (see below). This cell death program is induced under nutrient starvation and occurs simultaneously with the development of conidiophores. Thus, a brief description of the three types of cell death processes in fungi (see Suppl. Table 4) is appropriate at this point. 2.4.1. Autolysis Autolysis is the natural process of self-digestion of aged hyphae [101]. In this multistage process the cellular envelope becomes partially permeabilized as a consequence of hydrolase activity. Such permeabilization causes vacuolation and disruption of organelle and cell wall structures, generating a decline in biomass and liberation of the intracellular material to the culture/medium. Autolysis is a natural filamentous fungal survival mechanism that can be advanced or retarded by both intrinsic (fungal aging or hyphal differentiation) and extrinsic (nutrient limitation or other stress types) factors [101]. It is associated to an increase in extracellular proteolytic activities by secretion of glucanases and chitinases. This allows the recycling of products required to maintain hyphal growth in specific areas [102]. In A. nidulans a range of mutants have been isolated displaying an autolytic phenotype under specific growth conditions, being flbA- mutants the paradigm ([12]; see below). FlbA, as a regulator of the G protein FadA, seems to play a key role in the adequate balance among morphogenetic processes in A. nidulans, including autolysis. Another defined protein of this cell death process is the chitinase ChiB, playing a role in both intra and extracellular chitinase activities in flbA-induced autolysis [102]. In addition, autolysis-related chitinase and proteinase activities have been described to be induced through FluG, a key activator of asexual development [103]; see section 3.4.2). 2.4.2. Apoptosis Apoptosis is a type I PCD which is characterized by the following events (not in chronological order): 1) uptake of the apoptosis-inducing agent; 2) externalization of phosphatidylserine, normally located at the inner leaflet of the plasma membrane; 3) accumulation of DNA strand breaks; 4) release of cytochrome c from the mitochondrial intermembrane space to the cytosol; 5) involvement of cysteine proteases and 6) the presence of both mitochondriadependent and –independent signaling pathways, the former via homologues of the human apoptosis-inducing factor AIF [104]; references therein).


In A. nidulans the following are known apoptosis inducers: reactive oxygen species [105], farnesol [106], the Penicillium chrysogenum antifungal protein PAF [107] and sphingoid long-chain bases [108]. Savoldi et al. [106] proposed that farnesol induced the transcriptional accumulation of an AIF-like factor, aifA, and suggested that this mitochondrial oxidoreductase is required for decreasing the effects of farnesol and reactive oxygen species. In addition, farnesoldependent induction of apoptosis requires the poly(ADPribose)-polymerase (PARP; [109]), an enzyme involved in DNA-damage recognition and DNA repair. Interestingly, PARP also participates in correct conidia production [110] suggesting a link between apoptosis and asexual reproduction. 2.4.3. Autophagy Different cellular phenomena can be described by the term autophagy [111] but the few autophagy studies carried out to date in filamentous fungi focused exclusively on macroautophagy. This process is the non-specific engulfment of cytosolic components by double membrane vesicles which subsequently fuse with the vacuole/lysosome, where the contents are degraded [112]. Autophagy is a type II PCD linked to a nutrient recycling mechanism that may prolong cellular survival for differentiation and metal ion homeostasis. This was shown by the characterization of the downstream effectors of the TOR-kinase autophagy, atg, genes ([112]; references therein), such as the Aspergillus fumigatus protein Atg1 [113], whose homolog in A. nidulans, An1632.3, has not been characterized. Kiel and Klei [114] identified 34 putative Atg proteins in A. nidulans and suggested their additional role in pexophagy, an autophagy-related process responsible for the selective removal of peroxisomes from the cell. 3. ADAPTATIVE OR STRESS RESPONSE PATHWAYS The decision of activating or repressing the developmental programs described in the previous section is a combined response to a complex assortment of ambient stimuli, being most of them under two classes of stress: halo-osmotic and oxidative. As each stimulus is linked to a specific response mechanism, this section will list and describe the corresponding response pathways, discussing common regulatory elements that could participate in the integration of ambient signals into an endogenous positive or negative acting developmental signal. 3.1. The HogA Pathway This pathway is mainly required for osmotic and oxidative signal transduction. It is generally known that the exposition of cultures to the air induces development [115]. Some authors suggested that it occurs because alterations in reactive oxygen (and/or nitrogen) species concentration (ROS) may modulate vegetative growth and both sexual and asexual developments (for a general review in fungi, see [116]; for A. nidulans, see [117-119]). In addition, conidiation can be induced in liquid cultures or in aconidial mutants grown in solid media with elevated concentrations of phosphate or sodium salts [120]. Thus, the HogA pathway plays an impor-


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tant role in the regulation of development in response to these types of stress. Components from this pathway in S. pombe or S. cerevisiae are conserved in the A. nidulans proteome but several differences have been described (Suppl. Table 5) [121]. Extracellular signals are essentially sensed by two component systems based on Histidine Kinases (HK). Fifteen HKs coding genes have been identified in A. nidulans ([122]; and references therein) and some of them have been linked to development. TcsA, the homologue of the S. cerevisiae transmembrane osmosensor Sln1p, is required for conidiation [123] while the null mutant of its partner, tcsB, has no apparent morphological defects [124]. FphA is a phytochrome that plays a central role in the light sensing system, and also represses sexual reproduction and mycotoxin synthesis meanwhile induces asexual development [125]. NikA is a HK required for the response to fungicides as fludioxonil (phenylpyrrole-derivative that inhibits amino acid and carbohydrate uptake) and iprodione (dicarboximide-derivative that inhibit triglyceride biosynthesis), but it is also involved in osmotic adaptation and asexual development [126]. Recent work described the activation of phkA preferentially in cleistothecia [122]. The HK signal is transmitted to the unique histidinecontaining phosphotransmitter YpdA. In vitro experiments demonstrated that this signal can be subsequently transmitted to the homologue of the Skn7 response regulator (RR), SrrA [127]. Interestingly, a retrograde phosphorylation to FphA was also found, indicating the existence of a complex network of interactions. This network could compound responses to osmotic or oxidative signals with responses to light or hazardous molecules, for example, which bring about developmental processes. Thus, the group of fifteen HKs and, in general, the components of the phosphorelay system seem to be involved in sensing and transducing a wide range of ambient stimuli. The activities of the RRs SrrA and SskA, the next step in the pathway, are linked to oxidative and osmotic stress signal transduction, cell wall integrity, fungicide sensitivity and, finally, to asexual development [119]. Two additional RRs, SrrB and SrrC, were identified in A. nidulans, however, still remain to be clarified if they participate in the regulation of morphogenesis. Mechanistically, SskA transmits the signal to the MAP kinase cascade, formed by the MAP kinase kinase kinase SskB, the MAP kinase kinase PbsB and this to the MAP kinases HogA or MpkC [128]. hogA null mutant shows growth inhibition under high osmolarity, and a role for hogA in both asexual and sexual reproductive cycles has been described [128,129]. HogA activates the expression of the bZIP TF coding gene atfA [130], whose transcriptional activity is required for osmotic, oxidative and fungicide responses [131]. Besides, a parallel response route to HogA [132] is mediated by additional bZIP TFs belonging to the AP-1 family. AP-1 factors are required for the response to low concentrations of ROS and act in coordination with the CCAAT sequence-binding complex AnCF (or Hap complex; [133]). AP-1 and Atf1 homologues are considered as the two major activators of the proteins involved in the removal of the species that caused the stress. However, neither AtfA nor the A. nidulans AP-1


family protein NapA characterizations have reported a role in the regulation of asexual or sexual development [131,134]. 3.2. Prevention of ROS-Damage The response to endogenous or exogenous ROS is effected by a complex system including, on one hand, the enzymes superoxide dismutase (for superoxide dismutation to form hydrogen peroxide), catalases (decomposition of hydrogen peroxide) and perosidases (glutathione peroxidase and peroxiredoxins), and on the other hand, reducing mechanisms that include the pentose phosphate pathway, the thioredoxin and glutathione redox systems (Suppl. Table 6) [116]. However, it has also been shown that alterations in ROS levels caused by ROS producing enzymes, such as NADPH oxidases, Nox, play an essential role in different processes, including cell differentiation [135]. Semighini et al. [118] proposed a model in which localized Nox activity generated a pool of ROS defining a dominant polarity axis at the hyphal tip. Their genetic studies suggested a RacAdependent activation of Nox production. In addition, the putative p67phox regulatory subunit homolog, NoxR, is predicted to cooperate with Cdc42 in a parallel pathway regulating Nox localization at the cell [118]. Furthermore, the deletion of the putative p91phox homologue, noxA, blocks cleistothecia formation without any visible effect on asexual development [117]. The activity of the catalase CatB has been associated with growth and the start of conidiation [136]. CatA shows a spore-specific localization [137] while the catalaseperoxidase CatD/CpeA is induced in Hülle cells during sexual development [138]. In addition, the thioredoxin and glutathione systems interplay in the A. nidulans response to ROS [139]. It has been described that the thioredoxin A deletion mutant shows decreased growth, increased catalase activity and the inability to form both conidiophores and cleistothecia in the absence of oxidative stress [140]. All these observations support a link between ROS levels and cellular differentiation. However, the precise mechanism(s) by which the elements of this response pathway couple with the general regulators of morphogenetic pathways (vegetative, sexual and asexual) remains unknown. 3.3. The Light Response Regulatory System Oxidative stress response and developmental processes are conditioned by light in the genus Trichoderma [141]. In A. nidulans, light is a prerequisite for the switch from vegetative growth to sexual or asexual development and some elements of the light sensing machinery, as the HK FphA. also participate in the oxidative/salt-stress response pathway (see section 3.1 and below). Red light shifts the ratio of asexual to sexual structures towards asexual reproduction, displaying a maximum response at 680nm [142]. On the other hand, fruiting-body formation is favored in the dark [17]. Light also induces other processes as secondary metabolite [143] or manoprotein [144] biosynthesis. The light response is mainly based on the scaffold activity of the exclusively fungal protein, VeA (Suppl. Table 7) [145]. Deletion of the veA gene blocks sexual fruiting body formation while overexpression induces this process [146].

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Recent works have shown that VeA does not act as a light receptor or as a TF itself, although localizes in the nuclei in a light dependent manner [99,147]. Instead, VeA mediates interactions with a number of regulatory proteins through the VeA-complex [125,148,149]. VeA interacts with FphA, one of the 15 HKs identified in A. nidulans (see section 3.1), which acts as a phytochrome-like red-light receptor. FphA is involved in the repression of mycotoxin production and cleistothecia formation whereas it induces conidiation under red light conditions [150]. In addition, A. nidulans is able to sense blue light through the interaction of the White-Collar proteins LreA and LreB with VeA [125]. Both Lre proteins have antagonistic roles with respect to FphA. The C-terminal HK and RR domains of FphA interact with LreB [125]. Therefore, FphA could act as a repressor of LreA/LreB activity depending on the light source. Another well characterized VeA interactor is LaeA [151], a general regulator of secondary metabolism [151] and, consequently, of reproductive developmental pathways. VeA also interacts with other velvet-like proteins such as VelB, which in turn interacts with VosA, a regulator of late stages of asexual development involved in conidia maturation and trehalose biogenesis [78,148]. The VeA complex regulates the expression of a number of TFs involved in morphogenesis, as for example brlA [152], rosA, a negative regulator of sexual development [97] or ppoA, required for the synthesis of psiB oxylipin ([153]; see section 4). Other works have suggested a connection of the A. nidulans COP9 signalosome, a conserved multiprotein complex, in the regulation of eukaryotic development and the VeA light-dependent signal transduction pathway [154]. Overall, the light sensing machinery seems to integrate external stimuli such as light (through blue and red light receptors, LreA/B and FphA, respectively) or osmotic and oxidative levels of the media (through the HK domain of FphA). These signals could be transduced by VeA and modulated through its highly dynamic interactions with specific partners, regulating different developmental and biosynthetic pathways. 3.4. Primary Metabolism and Development Massive proteomic or transcriptomic approaches have identified several genes of A. nidulans primary metabolism whose expression is altered during osmo-adaptation or oxidative stress response [130,155]. Furthermore, it has been described that mutations in genes for primary metabolism often interfere with sexual or asexual development under conditions in which vegetative growth remains normal (see references in [12,17]). In addition, conidiation can also be induced in carbon or nitrogen-starved liquid cultures [156]. Nitrogen starvation generates normal conidiophores while the lack of a carbon source results in the production of phialide-like structures with short chains of conidia at the tip of hyphae. In this section, we will describe the relation of primary metabolism genes and the nutrient starvation pathway with development, the latter being the main activator pathway of conidiophore development. 3.4.1. Nitrogen Metabolism Nitric oxide and other derived reactive nitrogen species (RNS) are involved in cell signaling [116]. In A. nidulans,


several conidiation mutants isolated in early screens are affected in the urea cycle and, in general, in nitrogen metabolism (Suppl. Table 8) [157,158]. Probably, the paradigm of this class of mutants is the argB- mutant. ArgB is the ornithine transcarbamylase (OTC) enzyme of the arginine biosynthetic pathway. This trimeric enzyme participates in the urea cycle and catalyzes the reaction between carbamoyl phosphate and ornithine to form citruline and phosphate. Noteworthy, in animals, the synthesis of RNSs requires Larginine. Arginine catabolism has also a clinical interest, as in humans OTC deficiency results in an increase in alanine and glutamate levels, and also a subsequent elevation of ammonia concentration in tissues causing neurological disorders [159]. In A. nidulans, argB- mutations result in various developmental defects, both in asexual and sexual reproductive cycles [160]. In addition, our unpublished results show a phenotypic transformation of flbB and flbE aconidial mutants (see next section) in an argB- genetic background, producing numerous Hülle cells not associated to the formation of cleistothecia. The main regulator of nitrogen metabolism, AreA [161], is a GATA-type TF involved in the regulation of genes required for the utilization of nitrogen sources. Transcriptional regulation of areA is exerted by a complex mechanism whereby AreB, another GATA-type TF, acts as a negative regulator of AreA-dependent nitrogen catabolic gene expression under nitrogen-limiting or nitrogen-starvation growth conditions [162]. AreB is involved in polar growth, conidial germination and asexual development, though not in sexual development [162]. The link between nitrogen metabolism and sexual development in A. nidulans is supported by the phenotype of mutants at the amino acid biosynthetic pathways. Deletion of trpB, encoding for a tryptophan synthase, or hisB, participating in histidine biosynthesis, block cleistothecia production on medium poorly supplemented with tryptophan or histidine, respectively [163,164]. CpcA and CpcB are a bZIP type TF and a receptor for activated C-kinase 1 (the scaffold protein RACK1), respectively. These proteins participate in the control of amino-acid biosynthetic pathway, also called the cross-pathway network [165]. Both proteins seem to be mechanistically linked to sexual development since cpcA overexpression or cpcB deletion cause the same developmental block during cleistothecia production. These are some examples of connections between nitrogen metabolism and asexual/sexual developmental pathways, although the exact molecular mechanisms linking these processes remain unidentified. 3.4.2. Carbon Starvation and Cell Wall Integrity Pathway Carbon starvation in liquid media can induce the production of asexual phialide-like sterigmata bearing short chains of conidia [156,166]. Simultaneously to this differentiation phenomenon, an autolysis process has been described to occur, for the recycling of nutrients required for sporulation [103]. An efficient autolytic process requires the secretion of proteinases and chitinases, among other types of hydrolases (see section 2.4.1), and their up-regulation in these conditions depends on the regulatory activity of FluG-FlbA/E factors (Suppl. Table 9). Those commonly called Upstream De-


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velopmental Activators (UDA) form a seemingly complex network of linear and parallel signaling routes. Most of the UDA factors are TFs that co-ordinate autolysis, inhibition of vegetative growth and induction of brlA, resulting in the production of asexual structures [66,102]. UDA factors are being further characterized, allowing a deeper understanding of their role in the balance of developmental programs. Our recent work describes the involvement of UDAs in other morphological processes as branching of vegetative hyphae, cell wall integrity under osmotic stress or the synthesis of autoregulators ([66,167]; see section 4). The FluG factor blocks FadA-dependent vegetative growth signal by activating FlbA, the negative regulator of G protein signaling [12]. It would also remove the repressive effect of SfgA, a Gal4-type Zn(II)2Cys6 binuclear cluster TF [168], and simultaneously stimulate autolysis through elevated proteinase production [103]. FluG does not regulate directly the expression of downstream flb genes [167], but Flb factors can induce brlA expression as a consequence of the inhibition of the repression exerted by SfgA [168]. In the UDA pathway, FlbB arises as a general regulator of conidiation and other related processes. It interacts with FlbE at the Spitzenkörper of vegetative hyphae. This apical FlbB/FlbE complex may be involved in the transduction of environmental signals from the apex to nuclei [66]. In this sense, the conidiation signaling machinery could sense ambient conditions in vegetative hyphae to decide whether asexual development must be activated or not. In nuclei, FlbB induces the transcription of flbD, coding for a cMyb type TF [167]. Then, both proteins form a transcriptional complex that activates brlA expression, in a regulatory event that resembles developmental control mechanisms involving cMyb and bZIP counterparts in metazoans and plants. A question arising is whether the zinc finger type TF FlbC participates in the FlbB/FlbD complex. The brlA promoter is predicted to contain FlbC binding sequences [169] but our recent findings show that flbC deletion delays brlA expression without a total transcription inhibition, as occurs in flbB or flbD null mutants (Kwon, Yu, Ugalde, not published). In addition, the same study also suggests an additional role of FlbC during sexual development. 3.5. Calcium Signaling Calcium has a recognized effect on polar growth and conidiation in a wide collection of fungi (see for example, [170,171]). Most fungal hyphae exhibit a high calcium gradient at the tip [118]. Mutations in genes affecting calcium signaling alter growth and developmental programs in A. nidulans [172]. However, Ca+2-mediated signaling has been less investigated in filamentous fungi than in yeast [173] and, although recent works have added valuable information to this field, the machinery involved in calcium signaling and homeostasis in A. nidulans remains to be characterized in detail. The calcium response pathway comprises Ca+2 ATPases, channels and transporters, as well as the Ca+2/ Ca calmodulin/calcineurin/Crz1p signaling pathway (Suppl. Table 10) [32,174]. Most of the components of this pathway are as yet uncharacterized and among the known genes, only +2


a limited number of mutants show defects on sexual or asexual reproductive programs. In this sense, catalytic subunit A of the A. nidulans serine/threonine-specific protein phosphatase calcineurin has been newly characterized [172]. The null cnaA mutant exhibits severe morphological defects. Calcineurin activity was linked with asexual developmental induction, and found that the zinc finger TF CrzA supports this induction in a calcineurin and brlA-dependent manner. The crzA knock-out mutant displays less severe morphological defects than the phosphatase, however CrzA is required for normal growth and adaptation to osmotic stress [173,175]. A recent publication also supports the link between the calcium and osmotic/oxidative pathways in A. fumigatus [176]. Calmodulin (CaM) is a primary intracellular receptor participating in the Ca+2 homeostasis pathway and additionally regulating multiple cellular processes. CaM in A. nidulans is essential but the generation of a conditional alcA(p)::gfp::caM strain allowed Chen et al. [177] to characterize the protein. CaM activity is required during conidia germination and polar extension. The presence of CaM at the tip of growing metulae during conidiophore development suggests a putative role for calmodulin in the formation of those primary sterigmata. 3.6. pH Signaling A common feature of regulatory proteins required in calcium signaling, cation adaptation and homeostasis, as CrzA or the fungal specific C2H2-type zinc finger TF SltA, is that they are also required for resistance to alkaline pH [173]. Historically, the C2H2-type zinc finger PacC is the TF mediating control of gene expression in response to medium alkalinisation (Suppl. Table 10) [178]. Noticeably, its S. cerevisiae homologue, Rim101p, was first identified as a positive regulator of meiosis and sporulation [179]. Rim101p participates in alkaline pH-adaptation, invasive growth, ion tolerance and acquisition, and certain cell-wall characteristics [178]. In Candida albicans Rim101 controls pH-induced dimorphism whereby acidic conditions favor yeast-like growth and alkaline conditions favor hyphal-like growth [180]. In A. nidulans, mutations in pacC, leading to either strong loss-of-function or gain-of-function phenotypes, result in strong morphological defects of the colony, including compact growth and reduced conidia production compared to the wild type. However, no defects on sexual development were found [178]. The signal of ambient alkalinisation is transduced by the pal pathway, which is composed of six genes and their products, and lacks any phosphatase/kinase activity. In contrast to pacC mutants, two strains carrying the same or different mutant alleles in a pal gene cannot be meiotically crossed, suggesting their requirement in sexual development through a pathway not related with PacC (our unpublished observations). Overall, a role for the pH signaling pathway in development can be suggested considering, on one hand, the role of PacC homologues in yeast and dimorphic fungi and, on the other hand, the inability of mutants in this pathway for meiotic crosses. However, this speculative link requires further confirmation and deeper analyses.

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4. THE ROLE OF ENDOGENOUS MOLECULES: AUTOREGULATORS AND DEVELOPMENT The action of exogenous signals is complemented by other endogenous metabolites or autoregulators that are required for the induction and balance of developmental programs. In the past these signals have been referred to as autoinducers, autoinhibitors, quorum sensing factors or morphogens (revised in [181]). Autoregulators convey information of environmental conditions or the status of cells and regulate transitions between germination, vegetative growth, and asexual/sexual development [181]. Fungal respiration generates carbon dioxide, which is transformed into bicarbonate and protons after its reaction with water molecules [182]. Synthesis of endogenous bicarbonate induces progressive branching without a significant change in the specific growth rate at areas immediately behind the periphery of surface cultures [183]. At distal and central regions, increased branching was accompanied by progressive inhibition of the specific growth rate. Oxylipins are unsaturated fatty acids that play important roles in many biological systems as signal molecules [184]. These oxygenase-mediated oxygenated derivatives are produced by many organisms through the action of lipoxygenases. The eight-carbon oxylipins 1-octen-3-ol and 3octanone play distinct roles being the first the main inhibitor of germination and the latter an inducer of conidiation of A. nidulans aerated cultures grown in the dark. Its release to the medium requires functional forms of UDA factors and is repressed by VeA (Herrero-Garcia and Ugalde, in preparation). Furthermore, different papers described that mutants in the UDA pathway are unable to produce two metabolites required to induce asexual development [12,185]. The first metabolite has been associated with FluG activity and is equivalent to conidiogenone from Penicillium cyclopium, an extracellular diterpene required for conidiation ([186,187]; Rodriguez-Urra and Ugalde, unpublished). This metabolite would be accumulated after air exposure at the surface of vegetative hyphae above a threshold concentration, inhibiting SfgA and allowing conidiation induction (see section 3.4.2; [188]). The second metabolite may act in the same pathway downstream of the conidiogenone equivalent [185]. A third metabolite has been described to act in a parallel pathway and was associated with the activity of the transmembrane protein TmpA (Suppl. Table 11) [189]. The timing and balance between asexual and sexual structures, in addition to mycotoxin production, is modulated by the concentration and nature of oxylipins [190]. These are oleic()-, linoleic()- or linolenic()-derived unsaturated fatty acids called psi (precocious sexual inducer) factors. The synthesis pathway of psi factors is well established but, unfortunately, the molecular characterization of the metabolites associated to UDA activity and their production mechanisms remain largely unknown. Although the role of autoregulators in morphogenesis is proved, the elucidation of those structures is required to fully understand their role and the transition mechanisms between different morphogenetic programs of Aspergillus nidulans.


5. GENERAL OUTLOOK: AN INTEGRATED RESPONSE TO MULTIPLE ENVIRONMENTAL AND ENDOGENOUS CUES? The study of morphogenesis in the ascomycete Aspergillus nidulans has rendered many important contributions to our understanding of fungal development. However, pivotal questions on how the fungus integrates external signals into a unified response to coordinate the three main morphogenetic programs remain as yet unanswered. This review has attempted to update information available and propose possible speculative answers to those questions. The vegetative mycelium of a filamentous fungus like A. nidulans, actively growing at the edge of the colony, is able to immediately arrest growth in response to a vast assortment of abiotic and biotic stresses, and induce asexual or sexual reproductive cycles Fig. (2). Once the developmental decision has been adopted, the generation of various pseudohyphal modules ensues, and evidence shows that the program may be reverted at some stages of the process. For example, environmental changes as an increase in the availability of a nitrogen source can abort conidiophore development at advanced stages, provoking a reversion to hyphal growth modes. Etxebeste et al. [66] showed that an increase in nitrogen availability blocked phialide and conidia production, generating vegetative hyphae-like cells that elongated indefinitely. We predict that various points of conidiophore development will include checks for environmental cues and the associated mechanisms to revert or continue with the program. This versatile form of response requires for complex regulatory networks rather than linear sequential ones as postulated so far for the process. At the genetic level, wide “-omics” survey analyses are still pending but the emerging studies show that a considerable number of genes are implicated in the regulatory net-


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works, and future developments will help define those networks further. Thus far, the genes can be classified in two main groups. The first group includes those that code for proteins involved in the progression of development, participating in the spatio-temporal generation of the cell types associated to each sub-program module. The second group includes those involved in the transduction of external signals, consequently activating or repressing the required developmental programs. The synthesis of specific autoregulators may play a crucial role in governing the intra-colony responses and the fungal-fungal interactions. The elevated number of environmental determinants and genes involved contrast with the decision of activating/repressing reproduction. The convergence of such an array of signals and gene products into a major basic response is of evolutionary interest. This must be reflected at genetic and molecular levels, enabling the preexisting or newly developed mechanisms to integrate the different signals transduced by adaptative pathways in a unified signal to activate or repress the master genes of each developmental process. At this level, the HogA pathway may play a crucial role in the response to oxidative and osmotic stress, but also in the developmental response to light or nutrient starvation. Additional examples supporting this integrating point of view are the complexity of the conidiation master gene brlA locus, as well as the fact that the Spitzenkörper at the hypha apex incorporates factors (namely FlbB) involved in brlA activation. The existence of a regulatory network is also supported by the finding that various proteins which appear to play a role in vegetative hyphae also participate at specific stages of conidiophore development. The complexity of the light sensing and transducing mechanism exerted by the sophisticated VeA-complex or the involvement of factors like FlbA in the simultaneous induction and inhibition of conidiation and polar growth, respectively, are only two additional

Fig. (2). A scheme representing the transduction of environmental determinants into intracellular signals that control development. Environmental stimuli (left column) are, through stress response or adaptative pathways (second column), integrated into a unified intracellular signal (third column) to control (activate or repress) the master genes of each developmental program (right column).


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examples supporting the existence of a complex network of factors controlling development.

[17]

Recent studies point to the existence of a mix between democratic and hierarchical dynamics in the regulation of morphogenesis [191]. The former provides for versatility, while the latter provides order. It is possible that analysis of development in lower eukaryotes like the model organism Aspergillus nidulans may facilitate the understanding of the interplay between these two types of dynamics in response to stress.

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ACKNOWLEDGEMENTS This work was supported by the Spanish Ministerio de Educación y Ciencia through grant BFU2009-08701 to E.A.E, and by the Basque Government through grant IT39310 to U.U. O.E. is a contract researcher of The University of The Basque Country.

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SUPPLEMENTARY MATERIALS

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Supplementary material is available on the publishers Web site along with the published article.

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Received: May 20, 2010

Revised: June 30, 2010

Accepted: October 13, 2010

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