Functional and structural analysis of FST1 in Fusarium verticillioides

Fusarium verticillioides causes an important seed disease on maize and produces fumonisin B1 (FB1), a mycotoxin that is detrimental to human and animal health. Previous studies discovered that expression of FST1 is required for FB1 production and wild-type level of virulence on maize seeds. FST1 encodes a putative protein with 12 transmembrane domains with sequence similarity to hexose transporters. However, those studies have failed to prove its ability to transport glucose, fructose or mannose. I identified another three phenotypes associated with the lack of a functional FST1, which includes reduced hydrophobicity of hyphae, reduced macroconidia production, and increased sensitivity to hydrogen peroxide. My research compared the transcriptome of the wild type and strain Δfst1 when grown on autoclaved maize kernels. The 17 % of transcriptome (2677 genes) were differentially expressed. Examination of these genes indicated that the disruption of FST1 function affected genes involved in secondary metabolism, cell structure, conidiogenesis, virulence, and resistance to reactive oxygen species. Additionally, I used a Saccharomyces cerevisiae strain (Δitr1) lacking a functional inositol transporter gene (ITR1) to study the function of FST1. This yeast mutant grows poorly in myo-inositol medium and is not inhibited by FB1. I found that expression of FST1 in strain Δitr1 restored growth on myo-inositol medium and sensitivity to FB1 to levels observed in the wild-type yeast strain. The results indicate that FST1 can function as an inositol transporter and suggests it can transport FB1 into fungal cells. Finally, the functional importance of amino acids in FST1 was examined by creating targeted mutations in the central loop and C-terminus regions of the protein. Expression of these engineered FST1 genes in stain Δitr1 of S. cerevisiae and strain Δfst1 of F. verticillioidesindicated that both the central loop and C-terminus are critical for FST1 functionality. Overall this research has established the first characterized inositol transporter in filamentous fungi and has advanced our knowledge about the global regulatory functions of FST1