Lysine benzoylation (Kbz) is a newly identified post-translational modification, which participates in the regulation of a variety of cellular processes. However, the function of Kbz in pathogenic fungi remains unclear. Here, we first identified that FoxA is a benzoylated protein with two benzoylated sites at lysines 425 and 433. Mutations of Kbz sites in FoxA significantly reduced long-chain fatty acid (LCFA) utilization activity and exhibited a phenotype similar to that of foxA gene deletion mutant, including decreased conidiation and aflatoxin production, reduced seed colonization, and increased sclerotia formation. Metabolomic analyses indicated that the deletion of FoxA or interference with its benzoylation could disrupt peroxisomal β-oxidation, resulting in the accumulation of LCFAs. This disruption may inhibit conidiation and aflatoxin production by modulating the synthesis of 15d-PGJ(2)-G. Furthermore, we found that EsaA has benzoyltransferase activity in vitro and in vivo, and its expression influences the Kbz of FoxA and LCFA utilization activity. Notably, mutants at the acetylated sites exhibited phenotypes similar to those of the benzoylated site mutants, suggesting that acetylation also plays a significant role in FoxA protein. Our study uncovers a previously unknown mechanism by which benzoylation and acetylation regulate FoxA activity to affect the development, secondary metabolism, and pathogenicity of A. flavus.IMPORTANCEAs a predominantly plant-pathogenic fungus, Aspergillus flavus not only causes severe crop diseases and economic losses but also poses a global food safety threat due to its production of aflatoxins. Furthermore, under certain conditions, it can act as an opportunistic pathogen, endangering the health of both humans and animals. Numerous studies have shown that protein post-translational modifications, such as acetylation, succinylation, and benzylation, were involved in aflatoxin production; however, the exact mechanism was still unclear. This study reveals, for the first time, the molecular mechanism by which benzoylation regulates the functional execution of multifunctional β-oxidation hydratase/dehydrogenase FoxA proteins and further influences the development and aflatoxin synthesis in Aspergillus flavus. This discovery not only provides new insights for the prevention and control of aflatoxin contamination but also provides theoretical support for the study of secondary metabolism regulation mechanisms of other fungi.
Chen et al. (Mon,) studied this question.