Summary: Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The ΔFggcs1‐null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The ΔFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild‐type PH‐1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1‐mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host‐dependent.

Significance: The triterpenes are a large and highly diverse group of plant natural products. They are synthesized by cyclization of the linear isoprenoid 2,3-oxidosqualene into different triterpene scaffolds by enzymes known as triterpene synthases. This cyclization process is one of the most complex enzymatic reactions known and is only poorly understood. Here, we identify a conserved amino acid residue that is critical for both product and substrate specificity in triterpene synthases from diverse plant species. Our results shed new light on mechanisms of triterpene cyclization in plants and open up the possibility of manipulating both the nature of the precursor and product specificity, findings that can be exploited for the production of diverse and novel triterpenes.