Asexual Sporulation in A. nidulans
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Asexual Sporulation in A. nidulans

The signal transduction cascades regulating asexual sporulation in the Ascomycetous filamentous fungi A. nidulans (Aspergillus nidulans) can be divided into two phases: a Growth phase, in which cells become competent to respond to sporulation-inducing signals and an Asexual reproduction phase, including initiation of the Sporulation pathway and formation of spore-bearing structures. A. nidulans is homothallic (self-fertile). This fungus grows by forming an ordered network of filaments or hyphae that form a mycelium (Ref.1). The hyphae grow by apical extension and multiply by branching; giving rise to a radially symmetrical colony that expands at a constant rate. After a fixed period of time following vegetative growth, some hyphal cells within the center of the mycelium produce aerial branches that initiate asexual sporulation. This involves the formation of multicellular structures called Conidiophores that bear chains of spores called Conidia. Development requires a shift from highly polarized hyphal growth to growth by budding and transition from a multinucleate to a uninucleate state. These changes are directed by interplay between cell cycle regulators and developmental pathways. Conidia germinate when they contact a nutrient-rich medium, giving rise to hyphae and completing the cycle (Ref.2 & 3).

A number of physiological criteria regulate A. nidulans asexual development. However the signals that induce A. nidulans differentiation are not well defined. Under normal growth conditions, the only environmental requirement is exposure to air. This requirement for air does not appear to involve Oxygen or Carbondioxide levels, but rather results from changes in the cell surface induced by the abrupt formation of an air-water interface (Ref.4). Sporulation is also induced in hyphae grown in nutrient (Carbon or Nitrogen)-limiting conditions. However, nutritional stress plays a secondary role in induction of sporulation as nutrient limitation reduces conidiation of surface-grown colonies and continuous replacement of growth medium beneath a surface-grown colony does not repress conidiation. Conidiation is also dependent upon exposure of hyphae to red light. Induction of morphogenesis involves temporal and spatial regulation of several hundred genes, many of which function in Conidiophore assembly or Conidiospore differentiation (Ref.5).

Initiation of asexual sporulation in A. nidulans is governed by two parallel antagonistic pathways. The activation of the central pathway is dependent upon early regulatory gene products like, Flug (Protein-Flug), FlbA (Developmental Regulator-FlbA), FlbC (Putative Zinc Finger Protein) and FlbD (Ref.6). Although the isolation of the ECF (Extracellular Conidiation Factor) and the exact function of Flug remain elusive, the C-terminal region of Flug is required to induce sporulation. FlbA is the best candidate to be part of the signal transduction pathway regulated by the ECF. FlbA has a C-terminal RGS (Regulator of G-protein Signaling) domain that is shared by a family of proteins found in evolutionarily diverse organisms. RGS proteins negatively regulate G protein-mediated signaling pathways by activating the intrinsic GTPase activity of heterotrimeric FadA (Guanine Nucleotide-Binding Protein-Alpha Subunit). Flug is necessary for the synthesis of an ECF that initiates the asexual sporulation pathway by inducing the activities of FlbA, FlbC and FlbD, resulting in expression of BrlA (Bristle-A Protein/Regulatory Protein-BrlA), AbaA (Regulatory Protein-AbaA) and WetA (Regulatory Protein-WetA) proteins. Additionally, stimulation of FlbA causes inactivation of FadA, resulting in the inhibition of sporulation (Ref.7 & 8).

In addition to the sporulation pathway described above, a parallel pathway involving heterotrimeric G-protein, FadA regulates mycelium proliferation and antagonizes Conidiophore development. Primary role of the RGS domain protein FlbA in sporulation is to prevent activation of FadA by stimulating GTPase activity. FlbA, following activation by Flug, promotes asexual sporulation by inactivating FadA-dependent growth signaling. Initial mycelial growth is driven by activation of the G-Alpha protein FadA to its GTP-bound form. This pathway represses sporulation until the fungus gains developmental competence. The progression of sporulation is also influenced by BrlA-dependent induction of the activities of cell cycle kinases, NIMX/CDC2 (Cell Division Control Protein-2/Cyclin-Dependent Protein Kinase) and NIMA (Never in Mitosis/G2-Specific Protein Kinase-NIMA), promoting morphogenesis from the multinucleate, filamentous form to a uninucleate, budding form (Ref.9 & 10). Growth signal transduction mediated by FadA involves its activation by GDP-to-GTP exchange, presumably initiated by the binding of a Growth Factor ligand to a putative Gpr (G protein-Coupled Receptor). In addition to the FadA, mycelial proliferation is also in part regulated by SfaD (G-Protein-Beta Subunit). Flug positively regulates the production and secretion of an ECF that binds to a cell surface receptor, DsgA and induces the asexual sporulation pathway depending on the occurrence of two events: first, activation of FlbA by Flug to inhibit FadA-mediated growth signaling and second, activation by Flug of sporulation-specific functions dependent on the products of the FlbC, FlbD and BrlA genes (Ref.11).

The PKA (cAMP (Cyclic Adenosine 3,5-monophosphate)-Dependent Protein Kinase) in the filamentous fungus A. nidulans is a potential downstream target of FadA activity, in STC/ST (Sterigmatocystin) production and conidiation. The polyketide ST is a carcinogenic secondary metabolite produced by A. nidulans. Two of the genes required for ST production and Conidiation, FlbA and FadA, are involved in a G-protein signaling pathway. Aflr (Sterigmatocystin Biosynthesis Regulatory Protein), a ST biosynthesis pathway-specific transcription factor, and BrlA, encoding a conidiation-specific transcription factor, require FlbA and FadA for their normal expression. The molecular mechanisms connecting FlbA and FadA with Aflr and BrlA activity remain unknown. One logical candidate for transmitting a signal from FlbA and FadA to Aflr and BrlA is the cAMP-dependent PKA. The regulatory and catalytic subunits of PKA includes PKAR (PKA Regulatory Subunit/cAMP-Dependent Protein Kinase Regulatory Subunit) and PKAC (cAMP-Dependent Protein Kinase Catalytic Subunit), respectively (Ref.4). Thus FlbA-FadA signaling of asexual sporulation, ST production and vegetative growth is mediated only in part through PKA. The precise mechanisms by which extracellular signals are transduced to activate asexual development in A. nidulans still remain to be elucidated. Complete sequencing of the A. nidulans genome is expected to provide an invaluable resource for identifying additional components of the asexual sporulation pathway that may have been missed by conventional genetic screens for aconidial mutants. While MAPK (Mitogen-Activated Protein Kinase) cascades have not been implicated in asexual sporulation of A. nidulans, identification of FadA G-proteins suggests that a cAMP-dependent signaling cascade may be involved (Ref.12).