Both Colletotrichum and Magnaporthe spp. develop appressoria pigmented with melanin, which is essential for fungal pathogenicity. 1,8-Dihydroxynaphthalene (1,8-DHN) is believed to be polymerized to yield melanin around the appresorial cell wall through the oxidative activity of laccases. However, no 1,8-DHN laccase has yet been identified in either Colletotrichum or Magnaporthe spp. Here, we report a laccase gene, LAC2, that is involved in the appressorial melanization of Colletotrichum orbiculare, which causes cucumber anthracnose. LAC2 encodes a protein with a signal peptide and has high homology to fungal laccases. The conidial color of lac2 mutants is distinct from that of the C. orbiculare wild type, and the mutants are nonpathogenic. Notably, the mutant appressoria are defective in melanization, and a host invasion assay showed that the appressoria are nonfunctional. LAC2 was induced during appressorial melanization. These results suggest that LAC2 oxidizes 1,8-DHN in the appressoria. The LAC2 homologues of other fungi located in the same phylogenetic clade as LAC2 fully complemented the lac2 mutants. Interestingly, a LAC2 homologue, located in a different clade, complemented the conidial pigmentation but not appressorial melanization of the mutants, suggesting that the LAC2 function in appressorial melanization might only be conserved in laccases of the LAC2 clade.

To investigate effector gene regulation in the wheat pathogenic fungus Parastagonospora nodorum, the promoter and expression of Tox3 was characterised through a series of complementary approaches. Promoter deletion and DNase I footprinting experiments identified a 25 bp region in the Tox3 promoter as being required for transcription. Subsequent yeast one-hybrid analysis using the DNA sequence as bait identified that interacting partner as the C2H2 zinc finger transcription factor PnCon7, a putative master regulator of pathogenesis. Silencing of PnCon7 resulted in the down-regulation of Tox3 demonstrating that the transcription factor has a positive regulatory role on gene expression. Analysis of Tox3 expression in the PnCon7 silenced strains revealed a strong correlation with PnCon7 transcript levels, supportive of a direct regulatory role. Subsequent pathogenicity assays using PnCon7-silenced isolates revealed that the transcription factor was required for Tox3-mediated disease. The expression of two other necrotrophic effectors (ToxA and Tox1) was also affected but in a non-dose dependent manner suggesting that the regulatory role of PnCon7 on these genes was indirect. Collectively, these data have advanced our fundamental understanding of the Con7 master regulator of pathogenesis by demonstrating its positive regulatory role on the Tox3 effector in P. nodorum through direct interaction.