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The synthesis of phospholipids relies on a sort of genes, whose promoter regions contain inositol-sensitive upstream activation sequence (UAS INO ) and are regulated by the basic helix-loop-helix (bHLH)-type  ino2/ino4  transcription factor (TF) pair. Ten putative bHLH TFs have been found through whole genome sequencing of  Aspergillus niger , but none of these TFs have been characterized. In this study, we identified and characterized the bHLH-type TF  ino2(An02g04350)  in  A. niger.  Electrophoretic mobility shift assay (EMSA) and yeast two-hybrid assay demonstrated that  ino2  functions as a homodimer in UAS INO  genes (e.g.,  ino1  and  cho1 ) and binds to  opi1(An1g02370) in vitro . Real-time quantitative PCR of  ino1  and quantification of total phospholipid indicated that the  ino2  disruptant downregulated the transcription of  ino1  and the amount of total cellular phosphatidylinositol. In addition, phenotype analyses showed that a loss of  ino2  led to resistance to cell wall interference and DNA damage. Comparative transcriptome analyses showed that more than 1000 genes and GO terms associated with UAS INO , endoplasmic reticulum–associated protein degradation, phosphatidylinositol synthesis, chitin synthesis, and fatty acid synthesis were differentially expressed in Δino2 compared to the wild type (WT). Taken together, these observations indicate that the bHLH TF  ino2  functions as a homodimer that regulates the synthesis of phosphatidylinositol, fatty acid, and chitin and influences the homeostasis of the endoplasmic reticulum membrane.

Identification and Characterization of a Novel Basic Helix-Loop-Helix Transcription Factor of Phospholipid Synthesis Regulation in Aspergillus niger