Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability
Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability
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DOI：
10.1128/aem.01393-18
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发表时间：
2018-10-01
影响因子：
4.4
通讯作者：
Soberon, Mario
中科院分区：
文献类型：
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作者：
Gomez, Isabel;Ocelotl, Josue;Soberon, Mario
苏云金芽孢杆菌Cry 1Ab和Cry 1Fa毒素是控制重要害虫的环境安全杀虫剂。草地贪夜蛾（Spodoptera frugiperda）是一种重要的玉米害虫，其对Cry 1A毒素的敏感性较低，而Cry 1Fa对该害虫具有高度活性，并用于转基因玉米中以防治草地贪夜蛾。草地贪夜蛾防治来自Cry 1Ab的结构域III的β 16区域已被证明参与与不同鳞翅目昆虫中的受体如碱性磷酸酶（ALP）或氨肽酶（APN）的相互作用。对Cry 1Ab β 16（（STLRVN 514）-S-509）的氨基酸进行丙氨酸扫描诱变，发现某些β 16突变（如N514 A）导致Cry 1Ab对S.对其他鳞翅目幼虫如天蛾幼虫的毒力没有影响。对N514进行了彻底诱变，结果表明Cry 1Ab N514 F、N514 H、N514 K、N514 L、N514 Q和N5145突变增加了对S的毒性。食虫类。在Cry 1Fa（N507 A）中构建相应的突变。野生型和突变型毒素（Cry 1Ab、Cry 1AbN 514 A、Cry 1AbN 514 F、Cry 1Fa和Cry 1FaN 507 A）对四种S.来自墨西哥和巴西的一个frugiperda种群显示，Cry 1AbN 514 A和Cry 1FaN 507 A一致地显示出对五种S. frugiperda种群相比之下，Cry 1AbN 514 F仅在两个S. frugiperda种群分析。突变体Cry 1AbN 514 A和Cry 1AbN 514 F对中肠蛋白酶处理表现出更大的稳定性。此外，对Cry 1Ab突变体的结合分析表明，毒性增加与刷状缘膜囊泡结合增加和S.重要性草地贪夜蛾（Spodoptera frugiperda）是南美和北美的主要玉米害虫，也是非洲不同国家的入侵害虫。然而，在转基因作物中表达的苏云金芽孢杆菌Cry 1A毒素对它的控制很差，该毒素有效地控制了其他鳞翅目害虫。相比之下，表达Cry 1Fa的玉米在控制S. frugiperda，尽管由于抗性进化，其有效性正在丧失。本文描述的一些Cry 1Ab结构域III突变体显示出对S.而对烟草天蛾的毒力没有损失。因此，这些Cry 1Ab突变体可以提供有用的工程毒素，其沿着其他Cry毒素，将可用于开发表达堆叠蛋白的转基因玉米以有效控制S.田间的草地害虫和其它鳞翅目害虫。
Bacillus thuringiensis Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. Spodoptera frugiperda is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for S. frugiperda control. The beta 16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab beta 16 ((STLRVN514)-S-509) revealed that certain beta 16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for S. frugiperda without affecting the toxicity for other lepidopteran larvae, such as Manduca sexta larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N5145 mutations increased the toxicity toward S. frugiperda. A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four S. frugiperda populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18- fold increased toxicity against four of five S. frugiperda populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the S. frugiperda populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for S. frugiperda ALP, APN, and cadherin receptors.IMPORTANCE Spodoptera frugiperda is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Bacillus thuringiensis Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of S. frugiperda, although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for S. frugiperda without loss of toxicity to Manduca sexta. Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of S. frugiperda and other lepidopteran pests in the field.