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 <i>Saccharomyces cerevisiae</i>. 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 <i>Cochliobolus heterostrophus</i> 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 <i>Candida albicans</i> strains, suggesting interference with ergosterol metabolism. <i>In planta</i>, FA exhibited dose-dependent antifungal activity, significantly reducing <i>C. heterostrophus</i> 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.IMPORTANCEFungal infections are a growing threat to human health and agriculture, with rising antifungal resistance limiting treatment options. In this study, we used a genome-wide screening approach to identify ferulic acid (FA), a naturally occurring compound found in plants, as a promising antifungal agent. FA targets the same cellular pathway as many current antifungal drugs and works especially well when combined with fluconazole, a commonly used treatment. Remarkably, FA is also effective against drug-resistant <i>Candida albicans</i> strains, offering hope for new ways to treat difficult infections. In addition to its medical potential, FA protects maize from fungal pathogens, highlighting its usefulness as a sustainable and environmentally friendly crop protectant. These results suggest that FA could be developed into a versatile antifungal agent with applications in both clinical and agricultural settings, helping address the urgent need for new strategies to overcome antifungal resistance.