Hierarchical transcription factor interaction network: RsrF mediates dual-layer regulation of...

The efficient production of plant polysaccharide-degrading enzymes in fungi is regulated by complex regulatory networks but the full interactome controlling this process remains poorly understood. Herein we systematically construct a transcription factor (TF)-TF interaction network that specifically coordinates cellulase and amylase biosynthesis in Penicillium oxalicum. A key finding was the identification of a novel regulatory cascade in which the TF, P. oxalicum raw starch-degrading enzyme regulator F (PoxRsrF), directly activates expression of the TF gene (PoxRsrC) by binding a core motif (5'-GTTT-3'), which requires arginine residue R33 in PoxRsrF. PoxRsrC is known to induce the expression of another regulatory gene (PoxRsrA). In addition, a parallel regulatory layer mediated by a direct physical interaction between PoxRsrF and PoxRsrA was discovered, which depends on S120, S121, and T448 in PoxRsrF and residue E7 in PoxRsrA. Site-directed mutagenesis of these critical residues in P. oxalicum significantly reduces amylase production. Together, our results elucidate a multi-layered regulatory framework integrating transcriptional cascades with protein-protein interactions.