Oier Etxebeste

Aspergillus nidulans asexual development: making the most of cellular modules

Asexual development in Aspergillus nidulans begins in superficial hyphae as the programmed emergence of successive pseudohyphal modules, collectively known as the conidiophore, and is completed by a layer of specialized cells (phialides) giving rise to chains of aerial spores. A discrete number of regulatory factors present in hyphae play different stage-specific roles in pseudohyphal modules, depending on their cellular localization and protein-protein interactions. Their multiple roles include the timely activation of a sporulation-specific pathway that governs phialide and spore formation. Such functional versatility provides for a new outlook on morphogenetic change and the ways we should study it.

Aspergillus nidulans FlbE is an upstream developmental activator of conidiation functionally associated with the putative transcription factor FlbB

Aspergillus nidulans switches from vegetative growth to conidiation when aerial hyphae make contact with the atmosphere, or are subjected to specific environmental stress. The activation of the central conidiation pathway led by the transcription factor brlA is a critical milestone in this morphogenetic transition. A number of upstream developmental activators (UDAs), expressed in vegetative cells, are required for this process to occur in conjunction with cessation of vegetative growth. Mutants affected in these factors remain aconidial (fluffy) with low brlA expression levels (flb). In this report, we describe FlbE as a UDA containing two conserved but hitherto uncharacterized domains, which functions in close association with putative transcription factor FlbB. Both UDAs are functionally interdependent, and colocalize at the hypha tip in an actin cytoskeleton‐dependent manner. Moreover, bimolecular fluorescence studies show that they physically interact in vivo. These findings add evidence in favour of the existence of a signalling complex at or near the Spitzenkörper as an important part of the machinery controlling the morphogenetic transition between vegetative growth and conidiation.

Transcriptional Changes in the Transition from Vegetative Cells to Asexual Development in the Model Fungus Aspergillus nidulans

ABSTRACT Morphogenesis encompasses programmed changes in gene expression that lead to the development of specialized cell types. In the model fungus Aspergillus nidulans, asexual development involves the formation of characteristic cell types, collectively known as the conidiophore. With the aim of determining the transcriptional changes that occur upon induction of asexual development, we have applied massive mRNA sequencing to compare the expression pattern of 19-h-old submerged vegetative cells (hyphae) with that of similar hyphae after exposure to the air for 5 h. We found that the expression of 2,222 (20.3%) of the predicted 10,943 A. nidulans transcripts was significantly modified after air exposure, 2,035 being downregulated and 187 upregulated. The activation during this transition of genes that belong specifically to the asexual developmental pathway was confirmed. Another remarkable quantitative change occurred in the expression of genes involved in carbon or nitrogen primary metabolism. Genes participating in polar growth or sexual development were transcriptionally repressed, as were those belonging to the HogA/SakA stress response mitogen-activated protein (MAP) kinase pathway. We also identified significant expression changes in several genes purportedly involved in redox balance, transmembrane transport, secondary metabolite production, or transcriptional regulation, mainly binuclear-zinc cluster transcription factors. Genes coding for these four activities were usually grouped in metabolic clusters, which may bring regulatory implications for the induction of asexual development. These results provide a blueprint for further stage-specific gene expression studies during conidiophore development.

Tip-to-nucleus migration dynamics of the asexual development regulator FlbB in vegetative cells

In Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip‐to‐nucleus communication was demonstrated by photo‐convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C‐terminus and the bZIP DNA‐binding domain in the N‐terminus. FlbE, a critical FlbB interactor, binds the bZIP domain. Furthermore, the absence of FlbE results in loss of tip localization but not nuclear accumulation. flbE deletion also abrogates transcriptional activity indicating that FlbB gains transcriptional competence from interactions with FlbE at the tip. Finally, a bipartite nuclear localization signal is required for nuclear localization of FlbB. Those motifs of FlbB may play various roles in the sequence of events necessary for the distribution and activation of this transcriptionally active developmental factor. The tip accumulation, FlbE‐dependent activation, transport and nuclear import sketch out a process of relaying an environmentally triggered signal from the tip to the nuclei. As the first known instance of transcription factor‐mediated tip‐to‐nucleus communication in filamentous fungi, this provides a general framework for analyses focused on elucidating the set of molecular mechanisms coupling apical signals to transcriptional events.

Apical control of conidiation in Aspergillus nidulans

The infection cycle of filamentous fungi consists of two main stages: invasion (growth) and dispersion (development). After the deposition of a spore on a host, germination, polar extension and branching of vegetative cells called hyphae allow a fast and efficient invasion. Under suboptimal conditions, genetic reprogramming of hyphae results in the generation of asexual spores, allowing dissemination to new hosts and the beginning of a new infection cycle. In the model filamentous fungus Aspergillus nidulans, asexual development or conidiation is induced by the upstream developmental activation (UDA) pathway. UDA proteins transduce signals from the tip, the polarity site of hyphae, to nuclei, where developmental programs are transcriptionally activated. The present review summarizes the current knowledge on this tip-to-nucleus communication mechanism, emphasizing its dependence on hyphal polarity. Future approaches to the topic will also be suggested, as stimulating elements contributing to the understanding of how apical signals are coupled with the transcriptional control of development and pathogenesis in filamentous fungi.

Neurons show the path: tip-to-nucleus communication in filamentous fungal development and pathogenesis

Multiple fungal species penetrate substrates and accomplish host invasion through the fast, permanent and unidirectional extension of filamentous cells known as hyphae. Polar growth of hyphae results, however, in a significant increase in the distance between the polarity site, which also receives the earliest information about ambient conditions, and nuclei, where adaptive responses are executed. Recent studies demonstrate that these long distances are overcome by signal transduction pathways which convey sensory information from the polarity site to nuclei, controlling development and pathogenesis. The present review compares the striking connections of the mechanisms for long-distance communication in hyphae with those from neurons, and discusses the importance of their study in order to understand invasion and dissemination processes of filamentous fungi, and design strategies for developmental control in the future.

GmcA Is a Putative Glucose-Methanol-Choline Oxidoreductase Required for the Induction of Asexual Development in Aspergillus nidulans

Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation.

Beyond Asexual Development: Modifications in the Gene Expression Profile Caused by the Absence of the Aspergillus nidulans Transcription Factor FlbB

In the model fungus Aspergillus nidulans, asexual development is induced from vegetative hyphae by a set of early regulators including the bZIP-type transcription factor FlbB. To determine the range of genes under the influence of the transcriptional activity of FlbB and to characterize their role in fungal development, we sequenced and compared the transcriptomes of a ΔflbB mutant and its isogenic wild-type strain at different developmental stages. Results confirmed the activating role of FlbB on downstream regulators of conidiation such as flbD and brlA. However, FlbB has additional functions beyond the induction of asexual development. Among the changes observed, absence of a functional FlbB caused induction of the dba cluster and synthesis of a secondary metabolite with bactericidal properties. In addition, a new transcriptional target of FlbB was unveiled, urdA, that codes for a putative transcription factor that represses premature sexual development. Taken together, our results indicate that the activators of asexual development simultaneously exert a role on other cellular functions, including an inhibitory effect on the sexual cycle, and reinforce the hypothesis that mutually exclusive metabolic and cellular patterns are associated with different morphogenetic programs.

Cytoplasmic dynamics of the general nuclear import machinery in apically growing syncytial cells.

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus.

Photo-convertible tagging for localization and dynamic analyses of low-expression proteins in filamentous fungi

Photo-convertible fluorescent proteins (PCFPs) undergo a dramatic change in their excitation and emission spectra upon irradiation at specific wavelengths, thus rendering a different color. Dendra2 is a commercially available PCFP used to track the redistribution of proteins within cellular compartments, their life-time or interactions. Before photo-conversion Dendra2 exhibits green fluorescence, which becomes red after irradiation with either UV or blue lights. Multiple studies including Dendra2 as a molecular tool have been described in eukaryotes but not in filamentous fungi. Here we present a method to tag low-expression proteins from the filamentous fungus Aspergillus nidulans with Dendra2 and track their cellular dynamics. The regulator of asexual development FlbB was selected as control, a transcription factor that is expressed at low levels and can be used as a marker for the tip and nuclei of vegetative hyphae. This control provided us with a visual way to confirm the functionality of our genomic and plasmid constructs, since a non-functional FlbB protein renders a block in development and a characteristic aconidial phenotype. Our protocol combines standardized cloning and transformation procedures with the use of a mercury lamp microscope to convert and follow Dendra2 within cells. Hence, we present a rapid, simple and inexpensive method that makes tracking analysis of proteins that present technical difficulties to be followed feasible in filamentous fungi.