Jae-Hyuk Yu

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.

The gprA and gprB genes encode putative G protein-coupled receptors required for self-fertilization in Aspergillus nidulans

The filamentous fungus Aspergillus nidulans possesses both asexual and sexual reproductive cycles. Sexual fruiting bodies (cleistothecia) can be formed in both homothallic (self) and heterothallic (outcross) conditions. In this study, we characterized two genes, gprA and gprB, that are predicted to encode putative G protein‐coupled receptors (GPCRs) similar to fungal pheromone receptors. Deletion (Δ) of gprA or gprB resulted in the production of a few small cleistothecia carrying a reduced number of ascospores, whereas ΔgprAΔgprB eliminated fruiting body formation in homothallic conditions. However, nullifying gprA and/or gprB did not affect vegetative growth, asexual sporulation, Hülle cell formation or even cleistothecia formation in outcross, indicating that GprA and GprB are specifically required for self‐fertilization. The gprA and gprB genes encode two transcripts and, for both genes, larger transcripts are detectable during vegetative growth and asexual development whereas smaller transcripts accumulate during sexual development. Upregulation of nsdD encoding a key sexual developmental activator resulted in the production of barren cleistothecia in the ΔgprAΔgprB mutant, suggesting that NsdD can partially rescue the developmental defects caused by deletion of GPCRs and that GprA/B‐mediated signalling may activate other genes necessary for maturation of cleistothecia and ascosporogenesis. Deletion of gprA and/or gprB suppressed growth defects caused by ΔgprD, implying that GprA/B function downstream of GprD‐mediated negative control of sexual development.

VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

Conservation of structure and function of the aflatoxin regulatory gene aflR from Aspergillus nidulans and A. flavus.

Under limiting growth conditions,Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to theAspergillus flavus andAspergillus parasiticus aflatoxin regulatory geneaflR. Deletion of theA. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. BecauseA. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined byA. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of theA. flavus aflR gene inA. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved betweenAspergillus spp. and thataflR expression is sufficient to activate genes in the ST pathway.

The nsdD gene encodes a putative GATA‐type transcription factor necessary for sexual development of Aspergillus nidulans

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA‐type transcription factor with the type IVb zinc finger DNA‐binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over‐expressed, sexual‐specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).

A novel regulator couples sporogenesis and trehalose biogenesis in Aspergillus nidulans.

Trehalose is a compatible osmolyte produced by bacteria, fungi, insects and plants to protect the integrity of cells against various environmental stresses. Spores, the reproductive, survival and infection bodies of fungi require high amounts of trehalose for long-term survival. Here, via a gain-of-function genetic screen, we identify the novel regulator VosA that couples the formation of spores and focal trehalose biogenesis in the model fungus Aspergillus nidulans. The vosA gene is expressed specifically during the formation of both sexual and asexual spores (conidia). Levels of vosA mRNA and protein are high in both types of spore. The deletion of vosA results in the lack of trehalose in spores, a rapid loss of the cytoplasm, organelles and viability of spores, and a dramatic reduction in tolerance of conidia to heat and oxidative stress. Moreover, the absence of vosA causes uncontrolled activation of asexual development, whereas the enhanced expression of vosA blocks sporulation, suggesting that VosA also functions in negative-feedback regulation of sporogenesis. VosA localizes in the nucleus of mature conidia and its C-terminal region contains a potential transcription activation domain, indicating that it may function as a transcription factor primarily controlling the late process of sporulation including trehalose biogenesis. VosA is conserved in most fungi and may define a new fungus-specific transcription factor family.

LaeA Control of Velvet Family Regulatory Proteins for Light-Dependent Development and Fungal Cell-Type Specificity

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators.

Genetic control of asexual sporulation in filamentous fungi.

Asexual sporulation (conidiation) in the ascomycetous filamentous fungi involves the formation of conidia, formed on specialized structures called conidiophores. Conidiation in filamentous fungi involves many common themes including spatial and temporal regulation of gene expression, specialized cellular differentiation, intra-/inter-cellular communications, and response to environmental factors. The commencement, progression and completion of conidiation are regulated by multiple positive and negative genetic elements that direct expression of genes required for proper vegetative growth and the assembly of the conidiophore and spore maturation. Light is one of the key environmental factors affecting conidiation. Developmental mechanisms in Aspergillus nidulans and Neurospora crassa have been intensively studied, leading to important outlines. Here, we summarize genetic control of conidiation including the light-responding mechanisms in the two model fungi.

Regulators of G‐protein signalling in Aspergillus nidulans: RgsA downregulates stress response and stimulates asexual sporulation through attenuation of GanB (Gα) signalling

Regulators of G‐protein signalling play a crucial role in controlling the degree of heterotrimeric G‐protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (ΔrgsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, ΔrgsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G‐protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by ΔrgsA were suppressed by deletion of ganB encoding a Gα subunit, indicating that the primary role of RgsA is to control negatively GanB‐mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS–Gα pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.

Upstream and Downstream Regulation of Asexual Development in Aspergillus fumigatus

ABSTRACT The opportunistic human pathogen Aspergillus fumigatus produces a large quantity of asexual spores (conidia), which are the primary agent causing invasive aspergillosis in immunocompromised patients. We investigated the mechanisms controlling asexual sporulation (conidiation) in A. fumigatus via examining functions of four key regulators, GpaA (Gα), AfFlbA (RGS), AfFluG, and AfBrlA, previously studied in Aspergillus nidulans. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA, and AfwetA throughout the life cycle of A. fumigatus revealed that, while transcripts of AfflbA and AffluG accumulate constantly, the latter two downstream developmental regulators are specifically expressed during conidiation. Both loss-of-function AfflbA and dominant activating GpaAQ204L mutations resulted in reduced conidiation with increased hyphal proliferation, indicating that GpaA signaling activates vegetative growth while inhibiting conidiation. As GpaA is the primary target of AfFlbA, the dominant interfering GpaAG203R mutation suppressed reduced conidiation caused by loss of AfflbA function. These results corroborate the hypothesis that functions of G proteins and RGSs are conserved in aspergilli. We then examined functions of the two major developmental activators AfFluG and AfBrlA. While deletion of AfbrlA eliminated conidiation completely, null mutation of AffluG did not cause severe alterations in A. fumigatus sporulation in air-exposed culture, implying that, whereas the two aspergilli may have a common key downstream developmental activator, upstream mechanisms activating brlA may be distinct. Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed expression of AfbrlA in synchronized developmental induction, indicating that these upstream regulators contribute to the proper progression of conidiation.