GPCR-mediated glucose sensing system regulates light-dependent fungal development and mycotoxin production.

Microorganisms sense environmental fluctuations in nutrients and light, coordinating their growth and development accordingly. Despite their critical roles in fungi, only a few G-protein coupled receptors (GPCRs) have been characterized. The Aspergillus nidulans genome encodes 86 putative GPCRs. Here, we characterise a carbon starvation-induced GPCR-mediated glucose sensing mechanism in A. nidulans. This includes two class V (gprH and gprI) and one class VII (gprM) GPCRs, which in response to glucose promote cAMP signalling, germination and hyphal growth, while negatively regulating sexual development in a light-dependent manner. We demonstrate that GprH regulates sexual development via influencing VeA activity, a key light-dependent regulator of fungal morphogenesis and secondary metabolism. We show that GprH and GprM are light-independent negative regulators of sterigmatocystin biosynthesis. Additionally, we reveal the epistatic interactions between the three GPCRs in regulating sexual development and sterigmatocystin production. In conclusion, GprH, GprM and GprI constitute a novel carbon starvation-induced glucose sensing mechanism that functions upstream of cAMP-PKA signalling to regulate fungal development and mycotoxin production.

The Transcriptional Regulator HbxA Governs Development, Secondary Metabolism, and Virulence in Aspergillus fumigatus.

Aspergillus fumigatus is the leading cause of invasive aspergillosis, which in immunocompromised patients results in a mortality rate as high as 90%. Earlier studies showed that HbxA is a global regulator in Aspergillus flavus affecting morphological development and secondary metabolism. Here, we determined its role in A. fumigatus, examining whether HbxA influences the regulation of asexual development, natural product biosynthesis, and virulence of this fungus. Our analysis demonstrated that removal of the hbxA gene caused a near-complete loss of conidial production in the mutant strain, as well as a slight reduction in colony growth. Other aspects of asexual development are affected, such as size and germination of conidia. Furthermore, we showed that in A. fumigatus, the loss of hbxA decreased the expression of the brlA central regulatory pathway involved in asexual development, as well as the expression of the "fluffy" genes flbB, flbD, and fluG HbxA was also found to regulate secondary metabolism, affecting the biosynthesis of multiple natural products, including fumigaclavines, fumiquinazolines, and chaetominine. In addition, using a neutropenic mouse infection model, hbxA was found to negatively impact the virulence of A. fumigatusIMPORTANCE The number of immunodepressed individuals is increasing, mainly due to the greater life expectancy in immunodepressed patients due to improvements in modern medical treatments. However, this population group is highly susceptible to invasive aspergillosis. This devastating illness, mainly caused by the fungus Aspergillus fumigatus, is associated with mortality rates reaching 90%. Treatment options for this disease are currently limited, and a better understanding of A. fumigatus genetic regulatory mechanisms is paramount for the design of new strategies to prevent or combat this infection. Our work provides new insight into the regulation of the development, metabolism, and virulence of this important opportunistic pathogen. The transcriptional regulatory gene hbxA has a profound effect on A. fumigatus biology, governing multiple aspects of conidial development. This is relevant since conidia are the main source of inoculum in Aspergillus infections. Importantly, hbxA also regulates the biosynthesis of secondary metabolites and the pathogenicity of this fungus.

The Aspergillus flavus rtfA Gene Regulates Plant and Animal Pathogenesis and Secondary Metabolism.

Aspergillus flavus is an opportunistic fungal plant and human pathogen and a producer of mycotoxins, including aflatoxin B1 (AFB1). As part of our ongoing studies to elucidate the biological functions of the A. flavusrtfA gene, we examined its role in the pathogenicity of both plant and animal model systems. rtfA encodes a putative RNA polymerase II (Pol II) transcription elongation factor previously characterized in Saccharomyces cerevisiae, Aspergillus nidulans, and Aspergillus fumigatus, where it was shown to regulate several important cellular processes, including morphogenesis and secondary metabolism. In addition, an initial study in A. flavus indicated that rtfA also influences development and production of AFB1; however, its effect on virulence is unknown. The current study reveals that the rtfA gene is indispensable for normal pathogenicity in plants when using peanut seed as an infection model, as well as in animals, as shown in the Galleria mellonella infection model. Interestingly, rtfA positively regulates several processes known to be necessary for successful fungal invasion and colonization of host tissue, such as adhesion to surfaces, protease and lipase activity, cell wall composition and integrity, and tolerance to oxidative stress. In addition, metabolomic analysis revealed that A. flavusrtfA affects the production of several secondary metabolites, including AFB1, aflatrem, leporins, aspirochlorine, ditryptophenaline, and aflavinines, supporting a role of rtfA as a global regulator of secondary metabolism. Heterologous complementation of an A. flavusrtfA deletion strain with rtfA homologs from A. nidulans or S. cerevisiae fully rescued the wild-type phenotype, indicating that these rtfA homologs are functionally conserved among these three species.IMPORTANCE In this study, the epigenetic global regulator rtfA, which encodes a putative RNA-Pol II transcription elongation factor-like protein, was characterized in the mycotoxigenic and opportunistic pathogen A. flavus Specifically, its involvement in A. flavus pathogenesis in plant and animal models was studied. Here, we show that rtfA positively regulates A. flavus virulence in both models. Furthermore, rtfA-dependent effects on factors necessary for successful invasion and colonization of host tissue by A. flavus were also assessed. Our study indicates that rtfA plays a role in A. flavus adherence to surfaces, hydrolytic activity, normal cell wall formation, and response to oxidative stress. This study also revealed a profound effect of rtfA on the metabolome of A. flavus, including the production of potent mycotoxins.

cpsA regulates mycotoxin production, morphogenesis and cell wall biosynthesis in the fungus Aspergillus nidulans.

The model fungus Aspergillus nidulans synthesizes numerous secondary metabolites, including sterigmatocystin (ST). The production of this toxin is positively controlled by the global regulator veA. In the absence of veA (ΔveA), ST biosynthesis is blocked. Previously, we performed random mutagenesis in a ΔveA strain and identified revertant mutants able to synthesize ST, among them RM1. Complementation of RM1 with a genomic library revealed that the mutation occurred in a gene designated as cpsA. While in the ΔveA genetic background cpsA deletion restores ST production, in a veA wild-type background absence of cpsA reduces and delays ST biosynthesis decreasing the expression of ST genes. Furthermore, cpsA is also necessary for the production of other secondary metabolites, including penicillin, affecting the expression of PN genes. In addition, cpsA is necessary for normal asexual and sexual development. Chemical and microscopy analyses revealed that CpsA is found in cytoplasmic vesicles and it is required for normal cell wall composition and integrity, affecting adhesion capacity and oxidative stress sensitivity. The conservation of cpsA in Ascomycetes suggests that cpsA homologs might have similar roles in other fungal species.

The 14-3-3 Protein Homolog ArtA Regulates Development and Secondary Metabolism in the Opportunistic Plant Pathogen Aspergillus flavus.

The opportunistic plant-pathogenic fungus Aspergillus flavus produces carcinogenic mycotoxins termed aflatoxins (AF). Aflatoxin contamination of agriculturally important crops, such as maize, peanut, sorghum, and tree nuts, is responsible for serious adverse health and economic impacts worldwide. In order to identify possible genetic targets to reduce AF contamination, we have characterized the artA gene, encoding a putative 14-3-3 homolog in A. flavus The artA deletion mutant presents a slight decrease in vegetative growth and alterations in morphological development and secondary metabolism. Specifically, artA affects conidiation, and this effect is influenced by the type of substrate and culture condition. In addition, normal levels of artA are required for sclerotial development. Importantly, artA negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. An increase in AF is also observed in seeds infected with the A. flavus strain lacking artA Furthermore, the expression of other secondary metabolite genes is also artA dependent, including genes in the cyclopiazonic acid (CPA) and ustiloxin gene clusters, in this agriculturally important fungus.IMPORTANCE In the current study, artA, which encodes a 14-3-3 homolog, was characterized in the agriculturally and medically important fungus Aspergillus flavus, specifically, its possible role governing sporulation, formation of resistant structures, and secondary metabolism. The highly conserved artA is necessary for normal fungal morphogenesis in an environment-dependent manner, affecting the balance between production of conidiophores and the formation of resistant structures that are necessary for the dissemination and survival of this opportunistic pathogen. This study reports a 14-3-3 protein affecting secondary metabolism in filamentous fungi. Importantly, artA regulates the biosynthesis of the potent carcinogenic compound aflatoxin B1 (AFB1) as well as the production of other secondary metabolites.

Examining the evolution of the regulatory circuit controlling secondary metabolism and development in the fungal genus Aspergillus.

Filamentous fungi produce diverse secondary metabolites (SMs) essential to their ecology and adaptation. Although each SM is typically produced by only a handful of species, global SM production is governed by widely conserved transcriptional regulators in conjunction with other cellular processes, such as development. We examined the interplay between the taxonomic narrowness of SM distribution and the broad conservation of global regulation of SM and development in Aspergillus, a diverse fungal genus whose members produce well-known SMs such as penicillin and gliotoxin. Evolutionary analysis of the 2,124 genes comprising the 262 SM pathways in four Aspergillus species showed that most SM pathways were species-specific, that the number of SM gene orthologs was significantly lower than that of orthologs in primary metabolism, and that the few conserved SM orthologs typically belonged to non-homologous SM pathways. RNA sequencing of two master transcriptional regulators of SM and development, veA and mtfA, showed that the effects of deletion of each gene, especially veA, on SM pathway regulation were similar in A. fumigatus and A. nidulans, even though the underlying genes and pathways regulated in each species differed. In contrast, examination of the role of these two regulators in development, where 94% of the underlying genes are conserved in both species showed that whereas the role of veA is conserved, mtfA regulates development in the homothallic A. nidulans but not in the heterothallic A. fumigatus. Thus, the regulation of these highly conserved developmental genes is divergent, whereas-despite minimal conservation of target genes and pathways-the global regulation of SM production is largely conserved. We suggest that the evolution of the transcriptional regulation of secondary metabolism in Aspergillus represents a novel type of regulatory circuit rewiring and hypothesize that it has been largely driven by the dramatic turnover of the target genes involved in the process.

rtfA, a putative RNA-Pol II transcription elongation factor gene, is necessary for normal morphological and chemical development in Aspergillus flavus.

The filamentous fungus Aspergillus flavus is an agriculturally important opportunistic plant pathogen that produces potent carcinogenic compounds called aflatoxins. We identified the A. flavus rtfA gene, the ortholog of rtf1 in Saccharomyces cerevisiae and rtfA in Aspergillus nidulans. Interestingly, rtfA has multiple cellular roles in this mycotoxin-producing fungus. In this study, we show that rtfA regulates conidiation. The rtfA deletion mutant presented smaller conidiophores with significantly reduced conidial production compared to the wild-type strain. The absence of rtfA also resulted in a significant decrease or lack of sclerotial production under conditions that allowed abundant production of these resistance structures in the wild type. Importantly, the deletion of rtfA notably reduced the production of aflatoxin B1, indicating that rtfA is a regulator of mycotoxin biosynthesis in A. flavus. In addition, the deletion rtfA also altered the production of several unknown secondary metabolites indicating a broader regulatory scope. Furthermore, our study revealed that rtfA controls the expression of the global regulators veA and laeA, which further influence morphogenesis and secondary metabolism in A. flavus.

Secondary metabolism and development is mediated by LlmF control of VeA subcellular localization in Aspergillus nidulans.

Secondary metabolism and development are linked in Aspergillus through the conserved regulatory velvet complex composed of VeA, VelB, and LaeA. The founding member of the velvet complex, VeA, shuttles between the cytoplasm and nucleus in response to alterations in light. Here we describe a new interaction partner of VeA identified through a reverse genetics screen looking for LaeA-like methyltransferases in Aspergillus nidulans. One of the putative LaeA-like methyltransferases identified, LlmF, is a negative regulator of sterigmatocystin production and sexual development. LlmF interacts directly with VeA and the repressive function of LlmF is mediated by influencing the localization of VeA, as over-expression of llmF decreases the nuclear to cytoplasmic ratio of VeA while deletion of llmF results in an increased nuclear accumulation of VeA. We show that the methyltransferase domain of LlmF is required for function; however, LlmF does not directly methylate VeA in vitro. This study identifies a new interaction partner for VeA and highlights the importance of cellular compartmentalization of VeA for regulation of development and secondary metabolism.

An Aspergillus flavus secondary metabolic gene cluster containing a hybrid PKS-NRPS is necessary for synthesis of the 2-pyridones, leporins.

The genome of the filamentous fungus, Aspergillus flavus, has been shown to harbor as many as 56 putative secondary metabolic gene clusters including the one responsible for production of the toxic and carcinogenic, polyketide synthase (PKS)-derived aflatoxins. Except for the production of aflatoxins, cyclopiazonic acid and several other metabolites the capability for metabolite production of most of these putative clusters is unknown. We investigated the regulation of expression of the PKS-non-ribosomal peptide synthetase (NRPS) containing cluster 23 and determined that it produces homologs of the known 2-pyridone leporin A. Inactivation and overexpression of a cluster 23 gene encoding a putative Zn(2)-Cys(6) transcription factor (AFLA_066900, lepE) resulted in downregulation of nine and up-regulation of 8, respectively, of the fifteen SMURF-predicted cluster 23 genes thus allowing delineation of the cluster. Overexpression of lepE (OE::lepE) resulted in transformants displaying orange-red pigmented hyphae. Mass spectral analysis of A. flavus OE::lepE extracts identified the known 2-pyridone metabolite, leporin B, as well as the previously unreported dehydroxy-precursor, leporin C. We provide strong evidence that leporin B forms a unique trimeric complex with iron, not found previously for other 2-pyridones. This iron complex demonstrated antiinsectan and antifeedant properties similar to those previously found for leporin A. The OE::lepE strain showed reduced levels of conidia and sclerotia suggesting that unscheduled leporin production affects fungal developmental programs.

The mtfA transcription factor gene controls morphogenesis, gliotoxin production, and virulence in the opportunistic human pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the leading causative agent of invasive aspergillosis (IA). The number of cases is on the rise, with mortality rates as high as 90% among immunocompromised patients. Molecular genetic studies in A. fumigatus could provide novel targets to potentially set the basis for antifungal therapies. In the current study, we investigated the role of the transcription factor gene mtfA in A. fumigatus. Our results revealed that mtfA plays a role in the growth and development of the fungus. Deletion or overexpression of mtfA leads to a slight reduction in colony growth, as well as a reduction in conidiation levels, in the overexpression strain compared to the wild-type strain. Furthermore, production of the secondary metabolite gliotoxin increased when mtfA was overexpressed, coinciding with an increase in the transcription levels of the gliotoxin genes gliZ and gliP with respect to the wild type. In addition, our study showed that mtfA is also necessary for normal protease activity in A. fumigatus; deletion of mtfA resulted in a reduction of protease activity compared to wild-type levels. Importantly, the absence of mtfA caused a decrease in virulence in the Galleria mellonella infection model, indicating that mtfA is necessary for A. fumigatus wild-type pathogenesis.

Coordinated and distinct functions of velvet proteins in Fusarium verticillioides.

Velvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogen Fusarium verticillioides, previous studies showed that the velvet protein F. verticillioides VE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteins F. verticillioides VelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene, F. verticillioides CAT2 (FvCAT2). Deletion of FvCAT2 resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom.

VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus.

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator veA, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus Aspergillus flavus. We found that the expression of A. flavus homolog genes involved in the HOG signaling pathway is regulated by veA. Deletion of veA resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of cat1 and trxB indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the cat1 promoter, where the absence of VeA results in abnormally stronger complex formation with reduced cat1 expression and more sensitivity to ROS in a veA deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the cat1 promoter. Our study also revealed that veA positively influences the expression of the transcription factor gene atfB and that normal formation of DNA-protein complexes in the cat1 promoter is dependent on AtfB.

Generation of complexity in fungal terpene biosynthesis: discovery of a multifunctional cytochrome P450 in the fumagillin pathway.

Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene ß-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.

Functional characterization of a veA-dependent polyketide synthase gene in Aspergillus flavus necessary for the synthesis of asparasone, a sclerotium-specific pigment.

The filamentous fungus, Aspergillus flavus, produces the toxic and carcinogenic, polyketide synthase (PKS)-derived family of secondary metabolites termed aflatoxins. While analysis of the A. flavus genome has identified many other PKSs capable of producing secondary metabolites, to date, only a few other metabolites have been identified. In the process of studying how the developmental regulator, VeA, affects A. flavus secondary metabolism we discovered that mutation of veA caused a dramatic down-regulation of transcription of a polyketide synthase gene belonging to cluster 27 and the loss of the ability of the fungi to produce sclerotia. Inactivation of the cluster 27 pks (pks27) resulted in formation of greyish-yellow sclerotia rather than the dark brown sclerotia normally produced by A. flavus while conidial pigmentation was unaffected. One metabolite produced by Pks27 was identified by thin layer chromatography and mass spectral analysis as the known anthraquinone, asparasone A. Sclerotia produced by pks27 mutants were significantly less resistant to insect predation than were the sclerotia produced by the wild-type and more susceptible to the deleterious effects of ultraviolet light and heat. Normal sclerotia were previously thought to be resistant to damage because of a process of melanization similar to that known for pigmentation of conidia. Our results show that the dark brown pigments in sclerotia derive from anthraquinones produced by Pks27 rather than from the typical tetrahydronapthalene melanin production pathway. To our knowledge this is the first report on the genes involved in the biosynthesis of pigments important for sclerotial survival.

The role of Aspergillus flavus veA in the production of extracellular proteins during growth on starch substrates.

The aflatoxin-producer and opportunistic plant pathogenic, filamentous fungus Aspergillus flavus is responsible for the contamination of corn and other important agricultural commodities. In order to obtain nutrients from the host A. flavus produces a variety of extracellular hydrolytic enzymes. Interestingly, A. flavus amylase and protease activity are dependent on the global regulator veA, a gene known to regulate morphogenesis and secondary metabolism in numerous fungi. Analysis of starch degradation by fungal enzymes secreted into broths of starch- or corn kernel-based media showed a notable accumulation of glucose in samples of the A. flavus control strain while the deletion veA sample accumulated high levels of maltose and maltotriose and only a small amount of glucose. Furthermore, SDS-PAGE and proteomics analysis of culture broths from starch- or corn kernel-based media demonstrated differential production of a number of proteins that included a reduction in the amount of a glucoamylase protein in the veA mutant compared to the control strain, while an alpha-amylase was produced in greater quantities in the veA mutant. Quantitative real-time PCR and western blot analyses using anti-glucoamylase or alpha-amylase antisera supported the proteomics results. Additionally, an overall reduction in protease activity was observed in the veA mutant including production of the alkaline protease, oryzin, compared to the control strain. These findings contribute to our knowledge of mechanisms controlling production of hydrolases and other extracellular proteins during growth of A. flavus on natural starch-based substrates.

Role of the osaA Gene in Aspergillus fumigatus Development, Secondary Metabolism and Virulence.

Aspergillus fumigatus is the leading cause of aspergillosis, associated with high mortality rates, particularly in immunocompromised individuals. In search of novel genetic targets against aspergillosis, we studied the WOPR transcription factor OsaA. The deletion of the osaA gene resulted in colony growth reduction. Conidiation is also influenced by osaA; both osaA deletion and overexpression resulted in a decrease in spore production. Wild-type expression levels of osaA are necessary for the expression of the conidiation regulatory genes brlA, abaA, and wetA. In addition, osaA is necessary for normal cell wall integrity. Furthermore, the deletion of osaA resulted in a reduction in the ability of A. fumigatus to adhere to surfaces, decreased thermotolerance, as well as increased sensitivity to oxidative stress. Metabolomics analysis indicated that osaA deletion or overexpression led to alterations in the production of multiple secondary metabolites, including gliotoxin. This was accompanied by changes in the expression of genes in the corresponding secondary metabolite gene clusters. These effects could be, at least in part, due to the observed reduction in the expression levels of the veA and laeA global regulators when the osaA locus was altered. Importantly, our study shows that osaA is indispensable for virulence in both neutropenic and corticosteroid-immunosuppressed mouse models.

The fumagillin biosynthetic gene cluster in Aspergillus fumigatus encodes a cryptic terpene cyclase involved in the formation of ß-trans-bergamotene.

Fumagillin 1 is a meroterpenoid from Aspergillus fumigatus that is known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The genetic and molecular basis for biosynthesis of 1 had been an enigma despite the availability of the A. fumigatus genome sequence. Here, we report the identification and verification of the fma gene cluster, followed by characterization of the polyketide synthase and acyltransferase involved in biosynthesis of the dioic acid portion of 1. More significantly, we uncovered the elusive ß-trans-bergamotene synthase in A. fumigatus as a membrane-bound terpene cyclase.

veA-dependent RNA-pol II transcription elongation factor-like protein, RtfA, is associated with secondary metabolism and morphological development in Aspergillus nidulans.

In Aspergillus nidulans the global regulatory gene veA is necessary for the biosynthesis of several secondary metabolites, including the mycotoxin sterigmatocystin (ST). In order to identify additional veA-dependent genetic elements involved in regulating ST production, we performed a mutagenesis on a deletion veA (ΔveA) strain to obtain revertant mutants (RM) that regained the capability to produce toxin. Genetic analysis and molecular characterization of one of the revertant mutants, RM3, revealed that a point mutation occurred at the coding region of the rtfA gene, encoding a RNA-pol II transcription elongation factor-like protein, similar to Saccharomyces cerevisiae Rtf1. The A. nidulans rtfA gene product accumulates in nuclei. Deletion of rtfA gene in a ΔveA background restored mycotoxin production in a medium-dependent manner. rtfA also affects the production of other metabolites including penicillin. Biosynthesis of this antibiotic decreased in the absence of rtfA. Furthermore, rtfA is necessary for normal morphological development. Deletion of the rtfA gene in wild-type strains (veA+) resulted in a slight decrease in growth rate, drastic reduction in conidiation, and complete loss of sexual development. This is the first study of an Rtf1 like gene in filamentous fungi. We found rtfA putative orthologues extensively conserved in numerous fungal species.

Morphogenetic and developmental functions of the Aspergillus nidulans homologues of the yeast bud site selection proteins Bud4 and Axl2.

The yeast bud site selection system represents a paradigm for understanding how fungal cells regulate the formation of a polarity axis. In Saccharomyces cerevisiae, Bud4 and Axl2 are components of the axial bud site marker. To address the possibility that these proteins regulate cellular morphogenesis in filamentous fungi, we have characterized homologues of Bud4 and Axl2 in Aspergillus nidulans. Our results show that Bud4 is involved in septum formation in both hyphae and developing conidiophores. Whereas Axl2 appears to have no obvious role in hyphal growth, it is required for the regulation of phialide morphogenesis during conidiation. In particular, Axl2 localizes to the phialide-spore junction, where it appears to promote the recruitment of septins. Furthermore, the developmental regulators BrlA and AbaA control the expression of Axl2. Additional studies indicate that Axl2 is also involved in the regulation of sexual development, not only in A. nidulans, but also in the phylogenetically unrelated fungus Fusarium graminearum. Our results suggest that Axl2 plays a key role in phialide morphogenesis and/or function during conidiation in the aspergilli.

VeA regulates conidiation, gliotoxin production, and protease activity in the opportunistic human pathogen Aspergillus fumigatus.

Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality in immunocompromised patients. In this study, we show that veA, a major conserved regulatory gene that is unique to fungi, is necessary for normal morphogenesis in this medically relevant fungus. Although deletion of veA results in a strain with reduced conidiation, overexpression of this gene further reduced conidial production, indicating that veA has a major role as a regulator of development in A. fumigatus and that normal conidiation is only sustained in the presence of wild-type VeA levels. Furthermore, our studies revealed that veA is a positive regulator in the production of gliotoxin, a secondary metabolite known to be a virulent factor in A. fumigatus. Deletion of veA resulted in a reduction of gliotoxin production with respect to that of the wild-type control. This reduction in toxin coincided with a decrease in gliZ and gliP expression, which is necessary for gliotoxin biosynthesis. Interestingly, veA also influences protease activity in this organism. Specifically, deletion of veA resulted in a reduction of protease activity; this is the first report of a veA homolog with a role in controlling fungal hydrolytic activity. Although veA affects several cellular processes in A. fumigatus, pathogenicity studies in a neutropenic mouse infection model indicated that veA is dispensable for virulence.