Two transcription factors cooperatively regulate DHN melanin biosynthesis and development in Pestalotiopsis fici

Fungal 1,8-dihydroxynaphthalene (DHN) melanin plays important roles in UV protection, oxidative stress and pathogenesis. However, knowledge of the regulatory mechanisms of its biosynthesis is limited. Previous studies showed two transcription factors, PfmaF and PfmaH, located in the DHN melanin biosynthetic gene cluster (Pfma) in Pestalotiopsis fici. In this study, deletion of PfmaH resulted in loss of melanin and affected conidia cell wall integrity. Specifically, PfmaH directly regulates the expression of scytalone dehydratase, which catalyzes the transition of scytalone to T3HN. However, PfmaF disruption using CRISPR/Cas9 system affected neither DHN melanin distribution nor conidia cell wall integrity in P. fici. Unexpectedly, overexpression of PfmaF leads to heavy pigment accumulation in P. fici hyphae. Transcriptome and qRT-PCR analyses provide insight into the roles of PfmaF and PfmaH in DHN melanin regulation. PfmaH, as a pathway specific regulator, mainly regulates melanin biosynthesis that contributes to cell wall development. Furthermore, PfmaF functions as a broad regulator to stimulate PfmaH expression in melanin production, secondary metabolism as well as fungal development.