Genetic and Physical Interactions between Gα Subunits and Components of the Gβγ Dimer of Heterotrimeric G Proteins in Neurospora crassa

ABSTRACTHeterotrimeric G proteins are critical regulators of growth and asexual and sexual development in the filamentous fungusNeurospora crassa. Three Gα subunits (GNA-1, GNA-2, and GNA-3), one Gβ subunit (GNB-1), and one Gγ subunit (GNG-1) have been functionally characterized, but genetic epistasis relationships between Gβ and Gα subunit genes have not been determined. Physical association between GNB-1 and FLAG-tagged GNG-1 has been previously demonstrated by coimmunoprecipitation, but knowledge of the Gα binding partners for the Gβγ dimer is currently lacking. In this study, the threeN. crassaGα subunits are analyzed for genetic epistasis withgnb-1and for physical interaction with the Gβγ dimer. We created double mutants lacking one Gα gene andgnb-1and introduced constitutively active, GTPase-deficient alleles for each Gα gene into the Δgnb-1background. Genetic analysis revealed thatgna-3is epistatic tognb-1with regard to negative control of submerged conidiation.gnb-1is epistatic togna-2andgna-3for aerial hyphal height, whilegnb-1appears to act upstream ofgna-1andgna-2during aerial conidiation. None of the activated Gα alleles restored female fertility to Δgnb-1mutants, and thegna-3Q208Lallele inhibited formation of female reproductive structures, consistent with a need for Gα proteins to cycle through the inactive GDP-bound form for these processes. Coimmunoprecipitation experiments using extracts from thegng-1-FLAG strain demonstrated that the three Gα proteins interact with the Gβγ dimer. The finding that the Gβγ dimer interacts with all three Gα proteins is supported by epistasis betweengnb-1andgna-1,gna-2, andgna-3for at least one function.

Submerged Conidiation and Product Formation by Aspergillus niger at Low Specific Growth Rates Are Affected in Aerial Developmental Mutants

ABSTRACTExposure to an aerial environment or severe nutrient limitation induces asexual differentiation in filamentous fungi. Submerged cultivation ofAspergillus nigerin carbon- and energy-limited retentostat cultures both induces and fuels conidiation. Physiological and transcriptomic analyses have revealed that this differentiation strongly affects product formation. Since conidiation is inherent in the aerial environment, we hypothesized that product formation near zero growth can be influenced by affecting differentiation or development of aerial hyphae in general. To investigate this idea, three developmental mutants (ΔfwnA,scl-1, andscl-2mutants) that have no apparent vegetative growth defects were cultured in maltose-limited retentostat cultures. The secondary-metabolite profile of the wild-type strain defined flavasperone, aurasperone B, tensidol B, and two so far uncharacterized compounds as associated with conidium formation, while fumonisins B2, B4, and B6were characteristic of early response to nutrient limitation by the vegetative mycelium. The developmental mutants responded differently to the severe substrate limitation, which resulted in distinct profiles of growth and product formation.fwnAencodes the polyketide synthase responsible for melanin biosynthesis during aerial differentiation, and we show that conidial melanin synthesis in submerged retentostat cultures and aurasperone B production arefwnAdependent. Thescl-1andscl-2strains are two UV mutants generated in the ΔfwnAbackground that displayed reduced asexual conidiation and formed sclerotium-like structures on agar plates. The reduced conidiation phenotypes of thescl-1andscl-2strains are reflected in the retentostat cultivation and are accompanied by elimination or severely reduced accumulation of secondary metabolites and distinctly enhanced accumulation of extracellular protein. This investigation shows that submerged conidiation and product formation of a mitosporic fungus cultured at low specific growth rates can be fundamentally affected by interfering with the genetic program for differentiation of aerial hyphae, opening new perspectives for tailoring industrial performance.