Identification and characterization of genes regulating amylolysis and cellulolysis in the maize pathogen Fusarium verticillioides

Fusarium verticillioides is an important ear and stalk rot pathogen of maize. Understanding how F. verticillioides utilizes carbohydrates such as starch and cellulose will elucidate the genetic regulation of pathogenesis and could improve biomass conversion technologies required for biofuel production. The objective of this project was to identify genes in F. verticillioides that regulate hydrolysis of cellulose or amylose. The approach was to identify candidate genes through forward genetics and characterize their function through reverse genetics. To this end, I created a collection of insertional mutants via Restriction Enzyme Mediated Integration (REMI) with a novel cassette for promoter trapping (PT) and developed high-throughput screens to visualize hydrolysis of cellulose and amylose. Mutants that displayed enhanced or reduced hydrolysis of cellulose or amylose and contained a single copy of the REMI cassette were selected for further analysis. For each mutant, genome-walker Polymerase Chain Reaction (PCR) was used to define the insertion site and identify the disrupted gene. The PT cassette in REMI mutant ATK 1042 disrupted a gene predicted to encode an E3 ubiquitin ligase (designated UBL1). UBL1 was disrupted by targeted mutagenesis in the wild-type strain to confirm the phenotype of the ATK 1042 strain. Assessments of kernel colonization and mycotoxin biosynthesis indicate that UBL1 is involved in the regulation of starch hydrolysis and fumonisin B1 production. By creating a novel genetic screen to dissect the regulation of carbohydrate metabolism and identifying a previously uncharacterized gene involved in pathogenesis and mycotoxigenesis, this project significantly expanded the current understanding of kernel rot of maize caused by F. verticillioides.