1,8-Dihydroxynaphthalene Melanin Biosynthesis in <i>Colletotrichum fructicola</i> Is Developmentally Regulated and Requires the Cooperative Function of Two Putative Zinc Finger Transcription Factors

In ascomycetes, 1,8-dihydroxynaphthalene (DHN) melanin plays important protective functions and its production is usually coupled with development and environmental stress responses. The regulation of melanin biosynthesis, however, remains obscure. Colletotrichum fructicola is a phytopathogen with a broad host range that produces melanized appressoria and perithecia. In this study, we annotated melanin genes in a high-quality C. fructicola genome and characterized two zinc finger transcription factors (TFs) (cmr1 and cmr2) that form a loosely organized gene cluster with several melanin biosynthesis genes. Deleting either TF abolished melanization in both mycelia and perithecia but did not affect appressoria. The deletion mutants also showed perithecial development defects. Overexpressing cmr1 in Δcmr2 strongly activated the expression of melanin biosynthesis genes including pks1, scd1, t4hr1, and thr1 and caused hyper-accumulation of charcoal to black pigment(s). On the other hand, overexpressing cmr2 in Δcmr1 activated pks1, t4hr1, and thr1, but not scd1. We conclude that proper DHN melanin accumulation in C. fructicola requires the cooperative function of two in-cluster TFs that also regulate perithecial development. The study clarifies DHN melanin regulations in C. fructicola and expands the function of melanin in-cluster TFs to sex regulation.