Defining the Genetic Regulation of Appressorium Formation in Cercospora zeae-maydis

Cercospora zeae-maydis is one of the primary pathogens associated with gray leaf spot, one of the most damaging foliar diseases of maize in the world. Gray leaf spot can be managed to some extent by cultural practices and fungicide applications. To infect maize, C. zeae-maydis grows towards stomata and forms infectious structures, termed appressoria, over stomatal pores. Prior research on the pathogen revealed that appressorium formation is crucial for foliar infection. Although several genes involved in pathogenesis have been identified in C. zeae-maydis, the molecular regulation of appressorium formation in this pathogen is poorly understood. Specifically, how the fungus senses stomata and induces appressorium formation are unknown. The goal of this research was to elucidate the genetic regulation of pre-penetration infectious development in C. zeae-maydis. To identify genes involved in appressorium formation, a collection of 1409 genetically tagged random insertional mutants was generated via Agrobacterium tumefaciens-mediated transformation and assayed for defects relating to appressorium formation in vitro and in planta. Two mutants were identified that were defective in appressorium formation and pathogenicity on maize leaves. Target enrichment sequencing identified two genes that were disrupted in the mutants: a CAZyme gene of the GH76 family, and a mitogen activated kinase kinase kinase (MEKK) gene belonging to the STE11 family. Targeted deletion mutants of the MEKK-encoding gene failed to form appressoria on maize leaves and were apathogenic, thus confirming a role for the gene in pathogenesis. However, deletion mutants of the GH76 CAZyme formed appressoria and were pathogenic, which suggested that the mutation was not linked to the phenotype of interest. Thus, the key finding of this research implicated a specific MEKK pathway with the regulation of appressorium formation and pathogenesis in C. zeae-maydis.