Abstract
Heterotrimeric G-proteins play crucial roles in growth, asexual development, and pathogenicity of fungi. The regulator of G-protein signaling (RGS) proteins function as negative regulators of the G proteins to control the activities of GTPase in Gα subunits. In this study, we functionally characterized the six RGS proteins (i.e., RgsA, RgsB, RgsC, RgsD, RgsE, and FlbA) in the pathogenic fungus Aspergillus flavus. All the aforementioned RGS proteins were also found to be functionally different in conidiation, aflatoxin (AF) biosynthesis, and pathogenicity in A. flavus. Apart from FlbA, all other RGS proteins play a negative role in regulating both the synthesis of cyclic AMP (cAMP) and the activation of protein kinase A (PKA). Additionally, we also found that although RgsA and RgsE play a negative role in regulating the FadA-cAMP/PKA pathway, they function distinctly in aflatoxin biosynthesis. Similarly, RgsC is important for aflatoxin biosynthesis by negatively regulating the GanA-cAMP/PKA pathway. PkaA, which is the cAMP-dependent protein kinase catalytic subunit, also showed crucial influences on A. flavus phenotypes. Overall, our results demonstrated that RGS proteins play multiple roles in the development, pathogenicity, and AF biosynthesis in A. flavus through the regulation of Gα subunits and cAMP-PKA signals. IMPORTANCE RGS proteins, as crucial regulators of the G protein signaling pathway, are widely distributed in fungi, while little is known about their roles in Aspergillus flavus development and aflatoxin. In this study, we identified six RGS proteins in A. flavus and revealed that these proteins have important functions in the regulation of conidia, sclerotia, and aflatoxin formation. Our findings provide evidence that the RGS proteins function upstream of cAMP-PKA signaling by interacting with the Gα subunits (GanA and FadA). This study provides valuable information for controlling the contamination of A. flavus and mycotoxins produced by this fungus in pre- and postharvest of agricultural crops.