Jeong-Ah Seo

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.

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.

Functional analyses of heterotrimeric G protein Gα and Gβ subunits in Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae (anamorph: Fusarium graminearum) is a major toxigenic plant pathogen that causes head blight disease on small-grain cereals. The fungus produces the mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) in infected hosts, posing a threat to human and animal health. Despite its agricultural and toxicological importance, the molecular mechanisms underlying its growth, development and virulence remain largely unknown. To better understand such mechanisms, we studied the heterotrimeric G proteins of G. zeae, which are known to control crucial signalling pathways that regulate various cellular and developmental responses in fungi. Three putative Gα subunits, GzGPA1, GzGPA2 and GzGPA3, and one Gβ subunit, GzGPB1, were identified in the F. graminearum genome. Deletion of GzGPA1, a homologue of the Aspergillus nidulans Gα gene fadA, resulted in female sterility and enhanced DON and ZEA production, suggesting that GzGPA1 is required for normal sexual reproduction and repression of toxin biosynthesis. The production of DON and ZEA was also enhanced in the GzGPB1 mutant, suggesting that both Gα GzGPA1 and Gβ GzGPB1 negatively control mycotoxin production. Deletion of GzGPA2, which encodes a Gα protein similar to A. nidulans GanB, caused reduced pathogenicity and increased chitin accumulation in the cell wall, implying that GzGPA2 has multiple functions. Our study shows that G. zeae heterotrimeric G protein subunits can regulate vegetative growth, sexual development, toxin production and pathogenicity.

The pkaB Gene Encoding the Secondary Protein Kinase A Catalytic Subunit Has a Synthetic Lethal Interaction with pkaA and Plays Overlapping and Opposite Roles in Aspergillus nidulans

ABSTRACT Filamentous fungal genomes contain two distantly related cyclic AMP-dependent protein kinase A catalytic subunits (PKAs), but only one PKA is found to play a principal role. In Aspergillus nidulans, PkaA is the primary PKA that positively functions in vegetative growth and spore germination but negatively controls asexual sporulation and production of the mycotoxin sterigmatocystin. In this report, we present the identification and characterization of pkaB, encoding the secondary PKA in A. nidulans. Although deletion of pkaB alone does not cause any apparent phenotypic changes, the absence of both pkaB and pkaA is lethal, indicating that PkaB and PkaA are essential for viability of A. nidulans. Overexpression of pkaB enhances hyphal proliferation and rescues the growth defects caused by ΔpkaA, indicating that PkaB plays a role in vegetative growth signaling. However, unlike ΔpkaA, deletion of pkaB does not suppress the fluffy-autolytic phenotype resulting from ΔflbA. While upregulation of pkaB rescues the defects of spore germination resulting from ΔpkaA in the presence of glucose, overexpression of pkaB delays spore germination. Furthermore, upregulation of pkaB completely abolishes spore germination on medium lacking a carbon source. In addition, upregulation of pkaB enhances the level of submerged sporulation caused by ΔpkaA and reduces hyphal tolerance to oxidative stress. In conclusion, PkaB is the secondary PKA that has a synthetic lethal interaction with PkaA, and it plays an overlapping role in vegetative growth and spore germination in the presence of glucose but an opposite role in regulating asexual sporulation, germination in the absence of a carbon source, and oxidative stress responses in A. nidulans.

The Phosducin-Like Protein PhnA Is Required for Gβγ-Mediated Signaling for Vegetative Growth, Developmental Control, and Toxin Biosynthesis in Aspergillus nidulans

ABSTRACT Phosducin or phosducin-like protein (PhLP) is a positive regulator of Gβγ activity. The Gβ (SfaD) and Gγ (GpgA) subunits function in vegetative growth and developmental control in the model filamentous fungus Aspergillus nidulans. To better understand the nature of Gβγ-mediated signaling, phnA, encoding an A. nidulans PhLP, has been studied. Deletion of phnA resulted in phenotypes almost identical to those caused by deletion of sfaD, i.e., reduced biomass, asexual sporulation in liquid submerged culture, and defective fruiting body formation, suggesting that PhnA is necessary for Gβ function. The requirement for the RGS protein FlbA in asexual sporulation could be bypassed by the ΔphnA mutation, indicating that PhnA functions in FlbA-controlled vegetative growth signaling, primarily mediated by the heterotrimeric G protein composed of FadA (Gα), SfaD, and GpgA. However, whereas deletion of fadA restored both asexual sporulation and the production of sterigmatocystin (ST), deletion of sfaD, gpgA, or phnA failed to restore ST production in the ΔflbA mutant. Further studies revealed that SfaD, GpgA, and PhnA are necessary for the expression of aflR, encoding the transcriptional activator for the ST biosynthetic genes, and subsequent ST biosynthesis. Overexpression of aflR bypassed the need for SfaD in ST production, indicating that the results of SfaD-mediated signaling may include transcriptional activation of aflR. Potential differential roles of FadA, Gβγ, and FlbA in controlling ST biosynthesis are further discussed.

Natural occurrence of trichothecenes and zearalenone in Korean and imported beers

In order to survey the natural occurrence of Fusarium mycotoxins including deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) in beers consumed in Korea, a total of 54 extracts of Korean and imported beers were derivatized with heptafluorobutyric anhydride and analysed by gas chromatography-mass spectrometry with selected ion monitoring mode. DON was detected in 14 samples (26%) and NIV was detected in 43 samples (80%). ZEN was not detected in any of the samples tested. The incidence of DON was 19% for Korean beers and 50% for imported beers, whereas the incidence of NIV was 85% for Korean beers and 58% for imported beers. This is the first report that the beers consumed in Korea are contaminated with DON and NIV.