SummaryColletotrichum lagenarium and Magnaporthe grisea are plant pathogenic fungi that produce melanin during the appressorial differentiation stage of conidial germination and during the late stationary phase of mycelial growth. Here, we report the identification of genes for two unique transcription factors, CMR1 (Colletotrichum melanin regulation) and PIG1 (pigment of Magnaporthe), that are involved in melanin biosynthesis. Both Cmr1p and Pig1p contain two distinct DNA-binding motifs, a Cys2His2 zinc finger motif and a Zn(II)2Cys6 binuclear cluster motif. The presence of both these motifs in a single transcriptional regulatory protein is unique among known eukaryotic transcription factors. Deletion of CMR1 in C. lagenarium caused a defect in mycelial melanization, but not in appressorial melanization. Also, cmr1D mutants do not express the melanin biosynthetic structural genes SCD1 and THR1 during mycelial melanization, although the expression of these two genes was not affected during appressorial melanization.

We cloned three structural genes PKSI, SCDl and THRl encoding for major melanin biosynthetic enzymes and one regulatory gene CMRl of Colletotrichum lagenarium. Among them, SCD1 and THRI proteins were purified by heterologous expression system using E. coli and poly clonal antibodies against those proteins were prepared. Melanization of appressoria is developmentally regulated system; conidia, germ tubes and infection hyphae were not melanized during morphogenesis from conidia. The wild type strain 104-T of C. iagenarium shows temperature sensitivity for appressorium formation. Temporal transcriptional and translational patterns of melanin biosynthetic genes in appressorium-differentiating and non-differentiating conidia indicated that both transcriptional and post-transcriptional regulations were involved in the expression of melanin biosynthetic genes.