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.     

NsdD Is a Key Repressor of Asexual Development in Aspergillus nidulans

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (−) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (−) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.     

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.     

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.     

Additive roles of two TPS genes in trehalose synthesis,conidiation, multiple stress responses and host infection of a fungal insect pathogen

Intracellular trehalose accumulation is relevant to fungal life and pathogenicity. Trehalose-6-phosphate synthase (TPS) is known to control the first step of trehalose synthesis, but functions of multiple TPS genes in some filamentous fungi are variable. Here, we examined the functions of two TPS genes (tpsA and tpsB) in Beauveria bassiana, a fungal insect pathogen widely applied in arthropod pest control. Intracellular TPS activity and trehalose content decreased by 71–75 and 72–80% in ΔtpsA, and 21–30 and 15–45% in ΔtpsB, respectively, and to undetectable levels in ΔtpsAΔtpsB, under normal and stressful conditions. The three mutants lost 33, 50, and 98% of conidiation capacity in standard cultures. Conidial quality indicated by viability, density, intracellular trehalose content, cell wall integrity, and hydrophobicity was more impaired in ΔtpsA than in ΔtpsB and mostly in ΔtpsAΔtpsB, which was also most sensitive to nutritional, chemical, and environmental stresses and least virulent to Galleria mellonella larvae. Almost all of phenotypic defects in ΔtpsAΔtpsB approached to the sums of those observed in ΔtpsA and ΔtpsB and were restored by targeted gene complementation. Altogether, TpsA and TpsB play complementary roles in sustaining trehalose synthesis, conidiation capacity, conidial quality, multiple stress tolerance, and virulence, highlighting a significance of both for the fungal adaptation to environment and host.

     

Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter

Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.     

Roles of autophagy-related genes in conidiogenesis and blastospore formation,virulence, and stress response of Beauveria bassiana

Autophagy is a conserved intracellular recycling mechanism, in which autophagy-related genes 12 and 16 (ATG12 and ATG16) function in a complex controlling the ubiquitin-like conjugation system. In the insect-pathogenic fungus Beauveria bassiana, ATG12 and ATG16 were functionally characterized. Disruption of BbATG12 or BbATG16 resulted in the absence of autophagic bodies under starvation stress. ΔBbATG12 and ΔBbATG16 mutant strains displayed similar defects in asexual development (conidiation and blastospore formation) and tolerance to oxidative stress. ΔBbATG16 strain exhibited the impaired growth on the media with gelatin or chitin as a single nitrogen source, and ΔBbATG12 displayed decreased growth on the media with sucrose, fructose or maltose as a single carbon source. Both BbATG12 and BbATG16 were required for fungal virulence. BbATG16 mutation had more effects on fungal virulence than BbATG12 in topical infection assay, although both genes had similar contributions to fungal virulence in intrahemocoel injection assay. This study indicates that BbATG12 and BbATG16 mediate diverse biological functions in addition to their convergent roles in autophagy.     

Isolation and physical characterization of three essential conidiation genes from Aspergillus nidulans

We cloned and characterized three genes from Aspergillus nidulans, designated brlA, abaA and wetA, whose activities are required to complete different stages of conidiophore development. Inactivation of these genes causes major abnormalities in conidiophore morphology and prevents expression of many unrelated, developmentally regulated genes, without affecting expression of nonregulated genes. The three genes code for poly(A)⁺RNAs that begin to accumulate at different times during conidiation. The brlA-and abaA-encoded RNAs accumulate specifically in cells of the conidiophore. The wetA-encoded RNA accumulates in mature conidia. Inactivation of the brlA gene prevents expression of the abaA and wetA genes, whereas inactivation of the abaA gene prevents expression of the wetA gene. Our results confirm genetic predictions as to the temporal and spatial patterns of expression of these genes and demonstrate that these patterns are specified at the level of RNA accumulation.