The tetrameric pheromone module SteC‐MkkB‐MpkB‐SteD regulates asexual sporulation, sclerotia formation and aflatoxin production inAspergillus flavus

Abstract

For eukaryotes like fungi to regulate biological responses to environmental stimuli, various signalling cascades are utilized, like the highly conserved mitogen-activated protein kinase (MAPK) pathways. In the model fungus Aspergillus nidulans, a MAPK pathway known as the pheromone module regulates development and the production of secondary metabolites (SMs). This pathway consists five proteins, the three kinases SteC, MkkB and MpkB, the adaptor SteD and the scaffold HamE. In this study, homologs of these five pheromone module proteins have been identified in the plant and human pathogenic fungus Aspergillus flavus. We have shown that a tetrameric complex consisting of the three kinases and the SteD adaptor is assembled in this species. It was observed that this complex assembles in the cytoplasm and that MpkB translocates into the nucleus. Deletion of steC, mkkB, mpkB or steD results in abolishment of both asexual sporulation and sclerotia production. This complex is required for the positive regulation of aflatoxin production and negative regulation of various SMs, including leporin B and cyclopiazonic acid (CPA), likely via MpkB interactions in the nucleus. These data highlight the conservation of the pheromone module in Aspergillus species, signifying the importance of this pathway in regulating fungal development and secondary metabolism. K E Y W O R D S aflatoxin B1, Aspergillus flavus, pheromone module, sclerotia, secondary metabolism

The Pheromone Module SteC-MkkB-MpkB-SteD-HamE Regulates Development, Stress Responses and Secondary Metabolism in Aspergillus fumigatus

“…These plates were incubated in the presence of light for 4 days to induce asexual reproduction and production of conidia (Figure 2A). For each of the five mutant strains, a dramatic reduction in sporulation was observed, similar to what is observed in both A. nidulans (Bayram et al, 2012;Frawley et al, 2018) and A. flavus (Frawley et al, 2020), with the exception of the A. flavus hamE mutant, which did not show any defects in asexual reproduction. For the A. fumigatus mutants, the average values of conidia produced were expressed as a percentage of the CEA17 wild-type average, which was chosen to represent 100% production ( Figure 2C).…”

“…Using a genetic and proteomic approach, we detected physical interactions between these proteins. In combination with confocal imaging, these data suggest that these proteins form a MAP kinase pheromone module in the cytoplasm and that MpkB enters the nucleus, similar to what is observed in both A. nidulans (Bayram et al, 2012;Frawley et al, 2018) and A. flavus (Frawley et al, 2020). This work also provides evidence that the pheromone module is critical for the regulation of asexual sporulation, cell stress responses and secondary metabolism.…”

“…VeA activation results in assembly of the trimeric velvet complex (VeA-VelB-LaeA) which regulates secondary metabolism (Vallim et al, 2000;Atoui et al, 2008;Bayram et al, 2008Bayram et al, , 2012Sarikaya Bayram et al, 2010). Characterization of this pheromone module signaling pathway led to the identification of a homologous signaling module in the saprophytic fungus A. flavus (Frawley et al, 2020). This species is a prolific producer of the carcinogen aflatoxin B1 and causes contamination of a wide array of agricultural crops (Lewis et al, 2005;Yu et al, 2005;Amare and Keller, 2014;Rushing and Selim, 2019).…”

“…However, HamE was not shown to interact with the proteins of this pathway. This tetrameric complex was shown to assemble in the cytoplasm and is essential for the regulation of asexual sporulation, sclerotia formation and aflatoxin B1 production (Frawley et al, 2020).…”

“…Both secondary metabolism and various methods of fungal development are co-regulated via pheromone module signaling in A. nidulans (Bayram et al, 2012;Frawley et al, 2018) and A. flavus (Frawley et al, 2020). This information, coupled with the recent identification of the MpkB homolog in A. fumigatus (Manfiolli et al, 2019), led to the proposal that A. fumigatus may also utilize a similar mechanism of regulation to control its developmental programs and SM production.…”