Penicillium decumbens BrlA extensively regulates secondary metabolism and functionally associates with the expression of cellulase genes

Penicillium decumbens has been used in the industrial production of lignocellulolytic enzymes in China for more than 15 years. Conidiation is essential for most industrial fungi because conidia are used as starters in the first step of fermentation. To investigate the mechanism of conidiation in P. decumbens, we generated mutants defective in two central regulators of conidiation, FluG and BrlA. Deletion of fluG resulted in neither “fluffy” phenotype nor alteration in conidiation, indicating possible different upstream mechanisms activating brlA between P. decumbens and Aspergillus nidulans. Deletion of brlA completely blocked conidiation. Further investigation of brlA expression in different media (nutrient-rich or nutrient-poor) and different culture states (liquid or solid) showed that brlA expression is required but not sufficient for conidiation. The brlA deletion strain exhibited altered hyphal morphology with more branches. Genome-wide expression profiling identified BrlA-dependent genes in P. decumbens, including genes previously reported to be involved in conidiation as well as previously reported chitin synthase genes and acid protease gene (pepB). The expression levels of seven secondary metabolism gene clusters (from a total of 28 clusters) were drastically regulated in the brlA deletion strain, including a downregulated cluster putatively involved in the biosynthesis of the mycotoxins roquefortine C and meleagrin. In addition, the expression levels of most cellulase genes were upregulated in the brlA deletion strain detected by real-time quantitative PCR. The brlA deletion strain also exhibited an 89.1 % increase in cellulase activity compared with the wild-type strain. The results showed that BrlA in P. decumbens not only has a key role in regulating conidiation, but it also regulates secondary metabolism extensively as well as the expression of cellulase genes.     

The Putative Guanine Nucleotide Exchange Factor RicA Mediates Upstream Signaling for Growth and Development in Aspergillus

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.     

Deletion of the Aspergillus flavus Orthologue of A. nidulans fluG Reduces Conidiation and Promotes Production of Sclerotia but Does Not Abolish Aflatoxin Biosynthesis

The fluG gene is a member of a family of genes required for conidiation and sterigmatocystin production in Aspergillus nidulans. We examined the role of the Aspergillus flavus fluG orthologue in asexual development and aflatoxin biosynthesis. Deletion of fluG in A. flavus yielded strains with an approximately 3-fold reduction in conidiation but a 30-fold increase in sclerotial formation when grown on potato dextrose agar in the dark. The concurrent developmental changes suggest that A. flavus FluG exerts opposite effects on a mutual signaling pathway for both processes. The altered conidial development was in part attributable to delayed expression of brlA, a gene controlling conidiophore formation. Unlike the loss of sterigmatocystin production by A. nidulans fluG deletion strains, aflatoxin biosynthesis was not affected by the fluG deletion in A. flavus. In A. nidulans, FluG was recently found to be involved in the formation of dehydroaustinol, a component of a diffusible signal of conidiation. Coculturing experiments did not show a similar diffusible meroterpenoid secondary metabolite produced by A. flavus. These results suggest that the function of fluG and the signaling pathways related to conidiation are different in the two related aspergilli.     

Differential Roles of the ChiB Chitinase in Autolysis and Cell Death of Aspergillus nidulans

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (ΔflbA) mutant autolyzed completely after a 3-day incubation, the ΔflbA ΔchiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the ΔflbA-induced autolysis. However, importantly, both ΔflbA and ΔflbA ΔchiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the ΔflbA and ΔflbA ΔchiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, β-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.Autolysis can be described as enzymatic self-degradation of the cells. It involves the activation of several key enzyme classes, resulting in the catabolism of macromolecules within the cell (11, 12, 23). Autolysis is observed in most filamentous fungi at the late stages of cultures and is affected by aging, programmed cell death, development, nutrient limitation, and numerous other factors (39). Despite its fundamental importance in growth, differentiation, secondary metabolism, and heterologous protein production, autolysis is a poorly understood feature of fungal biology (26, 39). It is anticipated that the elucidation of the molecular mechanisms governing fungal autolysis would have great impacts on both fundamental and applied aspects of filamentous fungi.The Aspergillus nidulans ΔflbA mutant exhibits autolysis as a predominant phenotype (18). FlbA is a regulator of G protein signaling (RGS) that negatively controls vegetative growth signaling, primarily mediated by a heterotrimeric G protein composed of FadA (Gα) and SfaD::GpgA (Gβγ) (31, 33, 34, 42, 43). Loss of flbA function causes prolonged activation of G protein signaling, which results in uncontrolled proliferation of hyphal mass, the blockage of sporulation, and hyphal disintegration (autolysis). Because nearly the entire mycelium disappears during autolysis of ΔflbA mutant strains, some component of this phenomenon is hypothesized to involve enzymatic degradation as observed during autolysis of aging fungal cultures (11, 12, 23).In A. nidulans, the last step of autolysis is thought to be the degradation of the cell wall of empty hyphae, which is associated with increased proteinase and chitinase activities (10). There are 15 potential chitinase open reading frames (ORFs) in the genome of A. nidulans. Among these, the class V endochitinase ChiB was shown to play an important role in autolysis. The deletion of chiB considerably reduced the intracellular and extracellular chitinase activities, and the levels of ChiB significantly increased when the fungal cells were starved for carbon sources, an induced condition for hyphal autolysis of A. nidulans (41).In the present study we addressed two primary questions: (i) does ChiB function in the accelerated autolysis and/or cell death caused by loss of FlbA and (ii) are there other hydrolytic enzymes involved in autolysis and/or cell death in A. nidulans? In order to investigate a hypothetical connection between FlbA-controlled autolysis and the ChiB activity, we carried out genetic, biochemical, and cellular studies using the ΔchiB, ΔflbA, ΔflbA ΔchiB (double deletion), and chiB overexpression mutants. We found that, while ChiB plays a crucial role in ΔflbA-induced autolysis, ChiB is dispensable for cell death, corroborating the idea that cell death and autolysis are independent processes in A. nidulans (8). We further present the identification and partial functional characterization of four gene encoding the proteins accumulate at high level in ΔflbA and/or ΔflbA ΔchiB strains and propose a working model linking G protein signaling and autolysis.     

Overexpression of fIbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brIA and premature initiation of development

Aspergillus nidulans reproduces asexually by forming thousands of mitotically derived spores atop highly specialized multicellular organs termed conidiophores. We have identified a gene called flbA (for fluffy low brlA expression) that is required for initiation of A. nidulans conidiophore development. flbA mutants form abnormal colonies that have a distinct fluffy phenotype characterized by tightly interwoven aerial hyphae that autolyse as the colony matures. The requirement for fIbA in conidiophore development precedes activation of brlA, a primary regulator of conidiophore development. The wild-type flbA gene was isolated and found to encode a 3.0 kb mRNA that is expressed throughout the A. nidulans asexual life cycle. Overexpression of fIbA using an Inducible promoter resulted in misscheduled expression of brlA in vegetative ceils and caused hyphal tips to differentiate into spore-producing structures. Sequence analysis of a nearly full-length fIbA cDNA clone showed that fibA is predicted to encode a 717-amino-acid polypeptide with 30% identity to the Saccharomyces cerevisiae SST2 protein. SST2 is required by yeast cells for resuming growth following prolonged exposure to yeast mating pheromone and for mating partner discrimination. We propose that fIbA plays a related role in a signalling pathway for Aspergillus conidiophore development.     

Inducible RNA Interference of brlAbeta in Aspergillus nidulans

An inducible RNA interference (RNAi) construct composed of inverted repeating alcA promoters flanking the developmental regulatory gene brlAβ was tested in Aspergillus nidulans. On inducing medium, the RNAi strains failed to sporulate and lacked brlAα and brlAβ expression. RNAi was specific for brlAβ, but not brlAα, silencing, indicating brlAα regulation by brlAβ.     

Distinctive role of fluG in the adaptation of Beauveria bassiana to insect-pathogenic lifecycle and environmental stresses

The upstream developmental activation (UDA) pathway comprises three fluG-cored cascades (fluG-flbA, fluG-flbE/B/D and fluG-flbC) that activate the key gene brlA of central developmental pathway (CDP) to initiate conidiation in aspergilli. However, the core role of fluG remains poorly understood in other fungi. Here, we report distinctive role of fluG in the insect-pathogenic lifecycle of Beauveria bassiana. Disruption of fluG resulted in limited conidiation defect, which was mitigated with incubation time and associated with time-course up-regulation/down-regulation of all flb and CDP genes and another fluG-like gene (BBA_06309). In ΔfluG, increased sensitivities to various stresses correlated with repression of corresponding stress-responsive genes. Its virulence through normal cuticle infection was attenuated greatly due to blocked secretion of cuticle-degrading enzymes and delayed formation of hyphal bodies (blastospores) to accelerate proliferation in vivo and host death. In submerged ΔfluG cultures mimicking insect haemolymph, largely increased blastospore production concurred with drastic up-regulation of the CDP genes brlA and abaA, which was associated with earlier up-regulation of most flb genes in the cultures. Our results unveil an essentiality of fluG for fungal adaptation to insect-pathogenic lifecycle and suggest the other fluG-like gene to act as an alternative player in the UDA pathway of B. bassiana.     

FigA,a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae,Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca²⁺ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.     

Gβ-Like CpcB Plays a Crucial Role for Growth and Development of Aspergillus nidulans and Aspergillus fumigatus

Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented.     

8-Carbon oxylipins inhibit germination and growth, and stimulate aerial conidiation in Aspergillus nidulans

Germination of Aspergillus nidulans conidia in liquid cultures was progressively inhibited at inoculum loads above 1 × 10⁵ conidia mL^?1. High conidial densities also inhibited growth of neighbouring mycelia. The eight-carbon oxylipin 1-octen-3-ol was identified as the main inhibitor in a fraction also containing 3-octanone and 3-octanol.These three oxylipins also increased the conidiation rate of dark-grown surface cultures, but had no effect on liquid cultures. 3-octanone was the most conidiogenic compound. The action of 3-octanone required functional forms of developmental activators fluG, flbB-D and brlA, and was not additive to the conidiogenic effect of stress stimuli such as osmotic stress or carbon starvation.Oxylipins were produced shortly after hyphae made contact with the atmosphere and were most effective on aerial mycelia, indicating that they perform their signalling function in the gas phase.     

Spatial control of developmental regulatory genes inAspergillus nidulans

ThebrlA andabaA genes ofAspergillus nidulans regulate stages of conidiophore development and are themselves regulated during development.brlA⁻ mutants produce conidiophore stalks devoid of vesicles, sterigmata, and spores.abaA⁻ mutants produce most of the conidiophore structures but fail to form conidia. To assess the spatial expression of these two genes, we fused the 5′ flanking region ofbrlA orabaA to theEscherichia coli lacZ gene.A. nidulans transformants with a single copy of either fusion gene integrated at a defined heterologus locus (argB) expressedβ-galactosidase during conidiophore development, parallelingbrlA andabaA mRNA accumulation. Controls lacking the fusion genes produced little or noβ-galactosidase activity. A method forin situ detection ofβ-galactosidase was devised. Hyphae or conidiophores were permeabilized by treatment with chloroform vapors and stained with 5-bromo-4-chloroindolyl-β-d-galactoside.β-Galactosidase activity was detected in specific conidiophore cell types.brlA- andabaA-directedβ-galactosidase accumulated in vesicles, sterigmata, and immature conidia. This procedure should be applicable for determining cellular specificities of gene expression in fungi for which transformation systems exist.     

G-protein signaling mediates asexual development at 25 degrees C but has no effect on yeast-like growth at 37 degrees C in the dimorphic fungus Penicillium mameffei

The ascomycete Penicillium marneffei is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25°C, P. marneffei grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37°C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a Gα subunit of a heterotrimeric G protein from P. marneffei named gasA with high similarity to fadA in Aspergillus nidulans. Through the characterization of a ΔgasA strain and mutants carrying a dominant activating or a dominant interfering gasA allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating gasA mutant whose mutation results in a G42-to-R change (gasA^G42R) does not express brlA, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering gasA^G203R mutant shows inappropriate brlA expression and conidiation. Interestingly, the gasA mutants have no apparent defect in dimorphic switching or yeast-like growth at 37°C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.