Two homeobox transcription factors CgrHtf1 and CgrAfh1 hierarchically regulate asexual sporulation and appressorium formation in the maize anthracnose fungus Colletotrichum graminicola.

Maize anthracnose, caused by the fungal pathogen Colletotrichum graminicola, is among the most devastating diseases affecting maize production. Homeobox transcription factors (HTFs) regulate key developmental and physiological processes in eukaryotes, including fungal pathogenesis. In this study, we identified two HTFs, CgrHtf1 and CgrAfh1, in C. graminicola. Both CgrHtf1 and CgrAfh1 contain a conserved homeobox domain and exhibit distinct nuclear localization, consistent with their predicted roles as transcriptional regulators. Disruption of Cgrhtf1 sharply reduced conidial production while increasing melanin biosynthesis. In contrast, deletion of Cgrafh1 not only impaired conidiation but also abolished the formation of functional appressoria and hyphopodia. Notably, overexpression of Cgrafh1 enhanced appressorium formation compared to the wild-type strain, suggesting its crucial role in the morphogenesis of appressoria. Transcriptome analysis revealed that CgrHtf1 regulates many genes associated with melanin biosynthesis, fungal development and cell cycle control, while CgrAfh1 predominantly modulates the expression of genes linked to signal transduction, cell cycle progression and autophagy processes. Collectively, we demonstrate that CgrHtf1 controls conidiation and melanin biosynthesis, whereas CgrAfh1 governs appressorium development, revealing hierarchical regulation of infection-related morphogenesis in C. graminicola.