Su Xiaofeng, Rehman Latifur, Guo Huiming, Li Xiaokang, Cheng Hongmei
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.
Curr Genet. 2018 Feb;64(1):235-246. doi: 10.1007/s00294-017-0729-0. Epub 2017 Aug 10.
Verticillium dahliae is the most overwhelming plant pathogen, causing Verticillium wilt in a number of economic crops. The molecular mechanism is still unclear and identification of the genes involved in the pathogenicity or virulence of this fungus would benefit to uncover such mechanism. STT3 is a catalytic subunit of the multi-subunit oligosaccharyl transferase (OST) and plays an essential role in glycoprotein modification. Here, we characterized STT3 gene (VDAG_03232.1) of V. dahliae to explore its regulatory role in the development and virulence by deletion and complementation of this gene, as well as its silence in transgenic plants. The expression of the STT3 gene increased at the stage of conidia germination and reached its peak level with germ tube formation and elongation. We generated the knockout mutants (ΔSTT3) using protoplast transformation. Mycelial growth, sporulation ability and glycoprotein secretion were impaired when ΔSTT3 mutants were grown on media supplemented with different carbon sources. Moreover, ΔSTT3 mutants exhibited distinctly decreased germination ratio and reduction in virulence compared with the wild type (Vd wt) and complementary (ΔSTT3-C) strains. We also generated transgenic Nicotiana benthamiana (Trans-1 and -2) plants by expressing dsRNA against the STT3 gene. Transgenic plants showed significant reduction in the disease index and fungal biomass resulting in elevated resistance to V. dahliae compared with the wild-type plants when inoculated with Vd wt. Our results indicated that STT3 mediates the full virulence through the regulation in fungal development, hyphal growth, glycoprotein secretion of V. dahliae and merits further study as a potential RNAi target to control this fungus.
大丽轮枝菌是最具危害性的植物病原菌，可导致多种经济作物发生黄萎病。其分子机制仍不清楚，鉴定该真菌致病性或毒力相关基因将有助于揭示这一机制。STT3是多亚基寡糖基转移酶（OST）的催化亚基，在糖蛋白修饰中起关键作用。在此，我们对大丽轮枝菌的STT3基因（VDAG_03232.1）进行了表征，通过该基因的缺失、互补以及在转基因植物中的沉默来探究其在发育和毒力方面的调控作用。STT3基因的表达在分生孢子萌发阶段增加，并在芽管形成和伸长时达到峰值水平。我们利用原生质体转化法构建了基因敲除突变体（ΔSTT3）。当ΔSTT3突变体在添加不同碳源的培养基上生长时，其菌丝生长、产孢能力和糖蛋白分泌均受到损害。此外，与野生型（Vd wt）和互补（ΔSTT3-C）菌株相比，ΔSTT3突变体的萌发率明显降低，毒力也有所下降。我们还通过表达针对STT3基因的dsRNA构建了转基因本氏烟草（Trans-1和-2）植株。与野生型植株相比，转基因植株在接种Vd wt后，病情指数和真菌生物量显著降低，对大丽轮枝菌的抗性增强。我们的结果表明，STT3通过调节大丽轮枝菌的发育、菌丝生长和糖蛋白分泌来介导其完全毒力，作为控制该真菌的潜在RNAi靶点值得进一步研究。
