How menadione triggers aflatoxin synthesis in A. flavus

Aspergillus flavus is a saprophytic fungus responsible for world-wide spread harvest and post-harvest infections on cultivations, mainly on cereal grains and legumes. A. flavus is one of the main producers of aflatoxin B1, the most dangerous carcinogenic metabolite in nature. In relation to this, some ongoing climate changes favor plant susceptibility to the attack by this fungus with a consequent, dangerous increase of aflatoxins into previously unexploited feed and foodstuff. In order to address effectively the economic and sanitary consequences of A. flavus contamination, a detailed and extensive knowledge of the pathogen metabolism and of the environmental conditions triggering the different biological processes, is of paramount importance. In this study, we focus on the effects of oxidative stress in the intracellular compartment, obtained by adding menadione 0,1mM to the culture medium. Menadione is a chinone and a precursor of vitamin K, its cytotoxicity has been extensively investigated in several human and murine cellular lines as an intracellular ROS inductor. Chinones are very common in nature. As substrates for flavoenzymes they may incur in one electron reduction to semichinone which, conversely, reduce O2 to superoxide anion in the intracellular environment, therefore providing a stressing condition. We evaluate the response from A. flavus via several analytical approaches: mycelial growth, conidia quantification, aflatoxin B1 synthesis, antioxidant enzymes activity, intracellular ROS quantification. To evaluate gene expression, we exploit RNAseq technology for transcriptome analysis, plus RT-PCR of markers for cellular respiration, pentose phosphate pathway, and oxidative stress response and sirtuins expression. Lastly, we have extracted and evaluated oxylipins by an MRM based LC-MS/MS method, in order to ascertain if they may represent a more stable reactive signal able to trigger aflatoxin synthesis and conidiogenesis through a remodulation of A. flavus metabolism in oxidative stress conditions.