High resistance of Isaria fumosorosea to carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1) rather than tubulin mutation
High resistance of Isaria fumosorosea to carbendazim arises from the overexpression of an ATP-binding cassette transporter (ifT1) rather than tubulin mutation
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Isaria fumosorosea 对多菌灵的高抗性源于 ATP 结合盒转运蛋白 (ifT1) 的过度表达，而不是微管蛋白突变
DOI：
10.1111/j.1365-2672.2011.05188.x
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发表时间：
2012-01-01
影响因子：
4
通讯作者：
Feng, M. -G.
中科院分区：
文献类型：
--
作者：
Song, T. -T.;Ying, S. -H.;Feng, M. -G.
目的：探讨昆虫病原真菌烟孢Isaria对杀菌剂多菌灵产生耐药性的可能机制。方法与结果：从15株野生型菌株中筛选出1株多菌灵敏感菌株If116，经nano2诱变获得9株多菌灵抗性提高82 ~ 830倍，耐热性降低15 ~ 51%的突变体。比较从If116及其突变体的α -和β -微管蛋白基因推断的蛋白序列发现，尽管所有突变体的b-微管蛋白基因转录本水平是If116的0.87 - 7.16倍，但没有发现与增强抗性相关的可追踪位点突变。三个被检测的突变体表现出多药耐药性，因为它们在生长过程中对草铵膦、吡虫啉和其他六种杀菌剂的抗性明显高于If116。进一步检查罗丹明染色囊胚孢子发现存在药物外排泵蛋白(s)在所有耐多菌灵突变体。因此，我们分析了从野生型和突变株中克隆的一个atp结合盒（ABC）转运体基因（ifT1）及其启动子区域的序列。三个共同点突变分别位于Gal4、Abf1和Raf的结合位点，这三个位点是众多蛋白位点调控网络中至关重要的转录因子。这些点突变使所有突变体的ifT1表达量提高了17 - 137倍。结论：真菌对多菌灵等多种农药产生抗性的原因是ABC转运体在结合位点发生点突变引起的过表达。研究的意义和影响：首次在昆虫病原真菌中发现转运蛋白介导的多药耐药，在改善真菌杀虫剂与化学农药的相容性方面具有潜在的应用价值。
Aims: Probing possible mechanisms involved in the resistance of entomopathogenic fungus Isaria fumosorosea to carbendazim fungicide.Methods and Results: A carbendazim-sensitive strain (If116) selected from 15 wild-type strains was subjected to NaNO2-induced mutagenesis, yielding nine mutants with carbendazim resistance increased by 82- to 830-fold and thermo-tolerance decreased by 15-51%. Comparing the protein sequences deduced from the alpha-and beta-tubulin genes of If116 and its mutants revealed no traceable site mutation relating to the enhanced resistance although the transcripts levels of b-tubulin gene in all mutants were 0 87- to 7 16-fold of that in If116. Three examined mutants showed multidrug resistance because they were significantly more resistant to glufosinate, imidacloprid and other six fungicides than If116 during growth. Further examination of rhodamine-stained blastospores revealed existence of drug efflux pump protein(s) in all carbendazim-resistant mutants. Thus, the sequences of an ATP-binding cassette (ABC) transporter gene (ifT1) and its promoter region cloned from the wild-type and mutant strains were analysed. Three common point mutations were located, respectively, at the binding sites of Gal4, Abf1 and Raf, which are crucial transcription factors in the regulative network of numerous protein loci. Such point mutations elevated the ifT1 expression by 17 to 137-fold in all the mutants.Conclusions: The overexpression of the ABC transporter caused by the point mutations at the binding sites was responsible for the fungal resistance to various pesticides including carbendazim.Significance and Impact of Study: The transporter-mediated multidrug resistance found for the first time in entomopathogenic fungi is potential for use in improving mycoinsecticide compatibility with chemical pesticides.