Rehman Latifur, Su Xiaofeng, Li Xiaokang, Qi Xiliang, Guo Huiming, Cheng Hongmei
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Curr Genet. 2018 Jun;64(3):645-659. doi: 10.1007/s00294-017-0780-x. Epub 2017 Nov 25.
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 (ΔFreB) and complementary strains (ΔFreB-C) using protoplast transformation. When cultured on media supplemented with FeSO, FeCl and no iron, ΔFreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of ΔFreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in ΔFreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in ΔFreB. Compared with the Vd-wt and ΔFreB-C, ΔFreB-1 and ΔFreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). ΔFreB-1 and ΔFreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 µM HO was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, ΔFreB exhibited less disease symptoms than Vd-wt and ΔFreB-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.
铁还原酶是整合膜蛋白，参与将环境中的三价铁还原为生物可利用的二价铁。在最具危害性的植物病原真菌大丽轮枝菌中，这些铁还原酶尚未得到详细研究。在本研究中，我们通过原生质体转化构建敲除突变体（ΔFreB）和互补菌株（ΔFreB-C），探索了FreB基因（VDAG_06616）在大丽轮枝菌铁还原和毒力中的作用。当在补充有FeSO、FeCl且无铁的培养基上培养时，ΔFreB的生长和孢子产生显著降低，尤其是在无铁培养基上。与野生型菌株（Vd-wt）相比，ΔFreB的跨膜铁还原酶活性降低了50%。该活性在ΔFreB-C中完全恢复。同时，ΔFreB中其他相关基因（Frect-4、Frect-5、Frect-6和Met）的表达水平明显升高。与Vd-wt和ΔFreB-C相比，ΔFreB-1和ΔFreB-2在不同碳源（淀粉、蔗糖、半乳糖和木糖）上的菌落直径和孢子数量受损。平板扩散试验表明，当向真菌培养物中加入100 μM H₂O₂时，ΔFreB-1和ΔFreB-2对氧化应激也高度敏感。当接种本氏烟草植株时，ΔFreB表现出比Vd-wt和ΔFreB-C更少的病害症状。总之，本研究结果不仅表明FreB介导铁代谢，是大丽轮枝菌完全毒力所必需的，而且还将加速未来对该真菌致病机制的研究。
