Biological Integrated Pest Management Research Unit, Robert W. Holley Center for Agriculture and Health, USDA-ARS, 538 Tower Road, Ithaca, NY 14853, USA.
Curr Genet. 2012 Apr;58(2):105-16. doi: 10.1007/s00294-012-0368-4. Epub 2012 Feb 25.
Destruxins are among the most exhaustively researched secondary metabolites of entomopathogenic fungi, yet definitive evidence for their roles in pathogenicity and virulence has yet to be shown. To establish the genetic bases for the biosynthesis of this family of depsipeptides, we identified a 23,792-bp gene in Metarhizium robertsii ARSEF 2575 containing six complete nonribosomal peptide synthetase modules, with an N-methyltransferase domain in each of the last two modules. This domain arrangement is consistent with the positioning of the adjacent amino acids N-methyl-L: -valine and N-methyl-L: -alanine within the depsipeptide structure of destruxin. DXS expression levels in vitro and in vivo exhibited comparable patterns, beginning at low levels during the early growth phases and increasing with time. Targeted gene knockout using Agrobacterium-mediated transformation produced mutants that failed to synthesize destruxins, in comparison with wild type and ectopic control strains, indicating the involvement of this gene in destruxin biosynthesis. The destruxin synthetase (DXS) disruption mutant was as virulent as the control strain when conidial inoculum was topically applied to larvae of Spodoptera exigua, Galleria mellonella, and Tenebrio molitor indicating that destruxins are dispensable for virulence in these insect hosts. The DXS mutants exhibited no other detectable changes in morphology and development.
破坏素是昆虫病原真菌中研究最深入的次生代谢物之一，但尚未有明确的证据表明它们在致病性和毒力中的作用。为了确定这种肽的生物合成的遗传基础，我们在玫烟色棒束孢 ARSEF 2575 中鉴定了一个 23792bp 的基因，该基因包含六个完整的非核糖体肽合成酶模块，每个模块的最后两个模块中都有一个 N-甲基转移酶结构域。这种结构域排列与破坏素结构中相邻氨基酸 N-甲基-L：-缬氨酸和 N-甲基-L：-丙氨酸的定位一致。DXS 的体外和体内表达水平表现出相似的模式，在早期生长阶段开始时水平较低，随着时间的推移而增加。使用农杆菌介导的转化进行靶向基因敲除产生了无法合成破坏素的突变体，与野生型和异位对照菌株相比，表明该基因参与了破坏素的生物合成。与对照菌株相比，当将分生孢子接种物局部应用于小菜蛾、黄粉虫和黄粉虫幼虫时，破坏素合成酶 (DXS) 缺失突变体的毒力与对照菌株相同，表明破坏素在这些昆虫宿主中的毒力不是必需的。DXS 突变体在形态和发育方面没有表现出其他可检测到的变化。
