Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B₁ (FB₁) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB₁ biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB₁ biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB₁ biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.

Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rots. While stalk rot pathogens are prevalent worldwide, our understanding of stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified F. verticillioides FvSYN1 gene, which was demonstrated to plays an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of FvSyn1 protein motifs in F. verticillioides development and virulence. We generated FvSyn1 motif-specific deletion mutants to further investigate how different motifs contribute to development and virulence. Microscopic observation showed that Δfvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild type progenitor. Moreover, Δfvsyn1 mutant was sensitive to cell wall stress agents resulting in vegetative growth reduction. We showed that FvSyn1::GFP protein is associated with endomembrane but this outcome did not clarify why the deletion of this protein led to stress sensitivity and aberrant hyphal development. Characterization of FvSyn1 domains indicated that both Syntaxin N-terminus (SynN) domain and SNARE C-terminus domain play distinct roles in fungal development but collectively function in virulence. We also determined that two domains in FvSyn1 is not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 in hyphal growth, localization, cell wall stress response and virulence in F. verticillioides . Highlights: ► F. verticillioides SNARE protein FvSYN1 is important for vegetative growth and virulence. ► FvSYN1 deletion mutant performed better under cell wall stressors. ► Dissecting of two domains and investigate the roles.