Steinbach Denise, Gutbrod Oliver, Lümmen Peter, Matthiesen Svend, Schorn Corinna, Nauen Ralf
Bayer CropScience AG, R&D, Pest Control Biology, Monheim, Germany; Martin-Luther-University Halle-Wittenberg, Institute for Biology, Halle, Germany.
Bayer CropScience AG, R&D, Research Technologies, Monheim, Germany.
Insect Biochem Mol Biol. 2015 Aug;63:14-22. doi: 10.1016/j.ibmb.2015.05.001. Epub 2015 May 12.
Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where diamide insecticides largely failed to control diamondback moth populations. Thus, our study highlights the global importance of the G4946E RyR target-site mutation, which as a mechanism on its own, confers high-level resistance to diamide insecticides in diamondback moth.
邻氨基苯甲酰胺类和氟虫双酰胺属于一类新型杀虫剂，可作为昆虫兰尼碱受体（RyR）的构象敏感激活剂。这些化合物能防治多种不同的食草昆虫，包括小菜蛾，小菜蛾是十字花科作物上臭名昭著的全球害虫，最近由于小菜蛾RyR跨膜结构域中的靶位点突变而产生了抗性。在本研究中，我们进一步研究了我们最近在菲律宾小菜蛾品系（Sudlon）中鉴定出的RyR G4946E靶位点突变的遗传学和功能影响。Sudlon品系对G4946E突变是纯合的，并且在没有选择压力的实验室条件下已经维持了近四年，并且对所有商业邻氨基苯甲酰胺类杀虫剂仍表现出>2000倍的稳定抗性比率。其与敏感品系（BCS-S）进行正反交产生的F1后代没有显示出母系效应，并且表现出对邻氨基苯甲酰胺类敏感的表型，这表明其遗传方式几乎是常染色体隐性的。随后的回交表明Sudlon品系中邻氨基苯甲酰胺类抗性几乎是单基因性质。用小菜蛾胸肌微粒体膜制剂进行的放射性配体结合研究为RyR G4946E突变对邻氨基苯甲酰胺类特异性结合及其对[³H]兰尼碱结合的浓度依赖性调节的显著功能影响提供了直接证据。基于兔RyR1的冷冻电镜结构的计算模型表明，小菜蛾G4946E位于跨膜螺旋S4中，靠近假定参与电压传感器调节的S4-S5连接结构域，以及另一个最近描述的位于螺旋S2中的突变I4790M，与G4946E相对约13 Å。通过焦磷酸测序进行基因分型显示，在2013/14年从十个不同国家的地区采集的幼虫中存在RyR G4946E突变，在这些地区邻氨基苯甲酰胺类杀虫剂在很大程度上未能控制小菜蛾种群。因此，我们的研究突出了G4946E RyR靶位点突变的全球重要性，该突变本身作为一种机制，赋予小菜蛾对邻氨基苯甲酰胺类杀虫剂的高水平抗性。
