FreB is involved in the ferric metabolism and multiple pathogenicity-related traits of Verticillium dahliae

Ferric reductases are integral membrane proteins involved in the reduction of environmental ferric iron into the biologically available ferrous iron. In the most overwhelming phytopathogenic fungus, Verticillium dahliae, these ferric reductase are not studied in details. In this study we explored the role of FreB gene (VDAG_06616) in the ferric reduction and virulence of V. dahliae by generating the knockout mutants (Delta FreB) and complementary strains (Delta FreB-C) using protoplast transformation. When cultured on media supplemented with FeSO4, FeCl3 and no iron, Delta FreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of Delta FreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in Delta FreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in Delta FreB. Compared with the Vd-wt and Delta FreB-C, Delta FreB-1 and Delta FreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). Delta FreB-1 and Delta FreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 mu M H2O2 was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, Delta FreB exhibited less disease symptoms than Vd-wt and Delta FreB-C. In conclusion, the present findings not only indicate that FreB mediates the ferric metabolism and is required for the full virulence in V. dahliae, but would also accelerate future investigation to uncover the pathogenic mechanism of this fungus.