ChIP-SEQ of rpd3 and WT in Aspergillus flavus

Lysine lactylation (Kla) is a newly-identified post-translational modification (PTM) which is reported to play crucial roles in diverse biological processes in both prokaryotes and eukaryotes. However, its distribution, function and regulatory enzymes in fungus remain unknown. Through a proteomic approach, we identified a total of 3509 high-confidence Kla sites on 1083 proteins that were capable of undergoing lactylation in Aspergillus flavus (A. flavus). These lactylated proteins were highly enriched for metabolic functionality. We have identified Rpd3 as a delactylase both in vitro and in vivo. Loss of the gene Rpd3 affects growth, aflatoxin synthesis, and reactive oxygen species (ROS) homeostasis in A. flavus. Interestingly, we demonstrate that lactic acid promotes histone H3K27 lactylation, while Rpd3 erases this PTM both in vitro and intracellularly, regulating aflatoxin synthesis and ROS accumulation in A. flavus. We also performed RNA sequencing (RNA-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq) analysis and found that histones H3K27la can promote the gene expression of NADH oxidase family protein (NOX) associated with aflatoxin synthesis and ROS homeostasis. Additionally, our studies reveal that histone H3K27la can sense lactic acid stress, thereby mediating oxidative burst. Our identification of a lysine delactylase and functional network of Kla provide novel insight into the mechanisms globally regulating growth and aflatoxin synthesis in A. flavus.