Genome-Wide CRISPRi Screen and Proteomic Profiling Identify Key Genes Related to Ferulic Acid's Antifungal Activity
Fungal pathogens of plants must overcome host-imposed stressors, including antimicrobial small molecules. Ferulic acid (FA), a plant-derived phenolic compound, induces fungal stress and cell death. To uncover genetic determinants of FA sensitivity, we performed a genome-wide CRISPR interference (CRISPRi) screen in Saccharomyces cerevisiae. We confirmed that FA impairs yeast growth and triggers stress granule marker sequestration, establishing a relevant selection condition. The CRISPRi screen identified 194 genes involved in the FA-induced stress response and 12 whose repression enhanced resistance. Among them, ERG9, encoding squalene synthase, was most strongly enriched, and its repression conferred FA resistance alongside upregulation of HMG1, implicating the ergosterol biosynthesis pathway. Proteomic profiling of FA-resistant Cochliobolus heterostrophus strains further revealed conserved upregulation of ergosterol biosynthetic enzymes. FA also synergized with Fluconazole, a known ergosterol-targeting antifungal, and enhanced susceptibility in azole-resistant Candida albicans strains, suggesting interference with ergosterol metabolism. In planta, FA exhibited dose-dependent antifungal activity, significantly reducing C. heterostrophus lesion formation in maize. These findings establish FA as a promising antifungal agent that targets conserved lipid biosynthesis pathways and overcomes resistance mechanisms, supporting its potential as a sustainable therapeutic and agricultural fungicide.