Fus3 interacts with Gal83, revealing the MAPK crosstalk to Snf1/AMPK to regulate secondary metabolic substrates in filamentous fungi

The pheromone MAPK is essential for the vital activities of fungi and is widely identified in filamentous fungi of agricultural, medical, and industrial relevance. The targets have rarely been reported and it is difficult to understand the mechanism of pheromone MAPK signaling pathway. Aflatoxins (AFs), highly carcinogenic natural products, are produced by the secondary metabolism of fungi, such as Aspergillus flavus. Our previous studies demonstrated that Fus3 regulates AFs by modulating substrate levels in Aspergillus flavus, but no mechanism explain that in fungi. Here we show Gal83, a new target of Fus3, and identified the pheromone Fus3-MAPK signaling pathway regulates the Snf1/AMPK energy-sensing pathway to modulate aflatoxins synthesis substrates. In the screening for target proteins of Fus3, the Snf1/AMPK complexes {beta} subunit was identified by using tandem affinity purification and multi-omics, which physically interacted with Fus3 in vivo and vitro and received phosphorylation from Fus3. While neither aflatoxin transcript levels were down-regulated in gal83-mutant and fus3-mutant strains, significant decreases in aflatoxin B1, aflatoxin synthetic substrates levels and gene expression levels of primary metabolic enzymes were shown that both the Fus3-MAPK and Snf1/AMPK pathways could response energy signal. In conclusion, all the evidence unlocks a novel pathway of Fus3-MAPK to regulate AFs synthesis substrates by cross-talking to the Snf1/AMPK complexes. ImportanceAflatoxin poses a great threat to human and animal health and the economy, thus the mechanisms regulating aflatoxin synthesis have been of great interest. We have previously demonstrated that MAPK regulates aflatoxin biosynthesis significantly, but the regulatory mechanism of Fus3-MAPK is not clear. Here we found that Pheromone Fus3-MAPK responds to energy and transmits to Snf1/AMPK through phosphorylation, which regulates the level of secondary metabolic substrates in Aspergillus flavus, as a novel pathway of Fus3-MAPK. Fus3 interacts stably with Gal83 and colocalizes in the cytoplasm and nucleus, directly regulating the levels of aflatoxin synthetic substrates. These data advance our understanding of the regulation of aflatoxin by pheromone MAPK, and the mechanism of pheromone MAPK and Snf1/AMPK crosstalk regulation is confirmed. Overall, this has a positive effect on both fungal regulatory mechanisms and aflatoxin prevention and control.