Mechanistic insights into pregnenolone-mediated conidiation inhibition in Fusarium oxysporum

Fusarium oxysporum (FO) is a plant pathogen that causes significant economic losses worldwide. Conidia are the primary source of FO infection and survival till the next generation. Hence, controlling conidiation is an important goal in plant protection research. The Fusarium oxysporum LZ51 was isolated from infected tomato plants and identified through ITS sequencing. Shenzhen BGI Co., Ltd. performed RNA sequencing using the BGI Multiomics System. F. oxysporum genes were knocked out via homologous recombination. ERG2 and EBP GFP-tagged proteins were synthesized in E. coli BL21 (DE3). The expressed proteins were subjected to Microscale Thermophoresis (MST) analysis. The present study investigated the efficacy of pregnenolone as a conidiation inhibitor against Fusarium oxysporum. It was determined that pregnenolone (Preg) effectively inhibited conidiation of Fusarium oxysporum, with an inhibition rate of ~ 42% at 10 mg/L. Furthermore, RNA-seq analysis identified (5) upregulated genes in the steroid biosynthesis pathway, suggesting that pregnenolone regulates conidia production through this pathway. Knocking out the steroid biosynthesis genes ERG4, ERG2, FDFT1, EBP, and WOR2 significantly decreased conidial production. HPLC analysis showed significantly lower ergosterol content in all the FO mutants. Molecular docking results showed that ERG2 and EBP have higher binding affinity to pregnenolone as compared to other genes. Further analysis predicted that pregnenolone binds to both ERG2 and EBP. These findings validate ERG2 and EBP as molecular targets of pregnenolone, suggesting their roles in inhibiting fungal conidiation.