Resistance toBacillus thuringiensisCry1Ac toxin requires mutations in twoPlutella xylostellaATP-binding cassette transporter paralogs
Resistance toBacillus thuringiensisCry1Ac toxin requires mutations in twoPlutella xylostellaATP-binding cassette transporter paralogs
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DOI：
10.1371/journal.ppat.1008697
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发表时间：
2020-08-01
期刊：
影响因子：
6.7
通讯作者：
You, Minsheng
中科院分区：
文献类型：
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作者：
Liu, Zhaoxia;Fu, Shu;You, Minsheng
苏云金芽孢杆菌（Bt）叶面喷雾剂和表达Bt毒素的转基因作物被广泛用于控制害虫，但抗性的进化限制了它们的功效。多项研究表明，ATP结合盒（ABC）转运蛋白是重要的Bt受体，ABCC 2或ABCC 3的突变可导致Cry 1Ac毒素抗性，但这一过程尚不完全清楚。在这项研究中，我们应用正向和反向遗传分析，以证明高水平的Bt-Cry 1Ac抗性在小菜蛾需要同时在PxABCC 2和PxABCC 3突变。我们从一株小菜蛾Cry 1Ac抗性菌株（Cry 1 S1000）中发现了这两个基因的失活突变，并进行了遗传连锁分析，支持PxABCC 2和PxABCC 3是小菜蛾Cry 1Ac抗性的致病基因。然后，我们敲除了小菜蛾敏感参考菌株（G88）中的PxABCC 2和PxABCC 3，以证实高水平的Cry 1Ac抗性需要PxABCC 2和PxABCC 3的突变，而不是任何一个基因的突变。这一发现扩展了我们对复杂的Bt抗性过程的理解，并可能与其他鳞翅目昆虫中的Bt-Cry 1Ac抗性有关。小菜蛾（Plutella xylostella）是一种世界性害虫，也是第一个对来自革兰氏阳性细菌苏云金芽孢杆菌（Bt）的毒素产生田间抗性的物种。虽然先前的工作表明ATP结合盒转运子亚家族C2（ABCC 2）或C3（ABCC 3）基因的突变可以赋予Cry 1Ac抗性，但在这里，我们揭示了PxABCC 2和PxABCC 3的组合突变需要获得高水平的Cry 1Ac抗性，而不是简单的任一基因的突变。我们鉴定了在小菜蛾Cry 1Ac抗性菌株（Cry 1 S1000）中同时发生的PxABCC 2和PxABCC 3的自然突变，其中一个突变（R-A2）导致PxABCC 2的错误剪接，另一个突变（R-A3）导致PxABCC 3的过早终止。遗传连锁分析表明，R（A2）和R（A3）与Cry 1Ac抗性紧密连锁。R（A2）和R（A3）基因的导入使感病菌株G88获得了对Cry 1Ac的高抗性，证实了这些基因赋予抗性。为了进一步支持PxABCC 2和PxABCC 3在CrylAc抗性中的作用，将移码突变单独引入PxABCC 2和PxABCC 3中，并在G88菌株中与CRISPR/Cas9介导的诱变组合。基于CRISPR的突变菌株的生物测定，加上遗传互补测试，表明单独缺失PxABCC 2或PxABCC 3提供了< 4倍的Cry 1Ac耐受性，而两个基因一起破坏赋予了> 8，000倍的Cry 1Ac抗性，这表明PxABCC 2和PxABCC 3的冗余/互补作用。这一工作进一步加深了我们对小菜蛾Bt抗性的理解，证明了高水平的Cry 1Ac抗性需要PxABCC 2和PxABCC 3基因的突变。
Author summary Bacillus thuringiensis(Bt) foliar sprays and transgenic crops expressing Bt toxins are used extensively to control insect pests, but the evolution of resistance limits their efficacy. Multiple studies have reported that ATP-binding cassette (ABC) transporters are important Bt receptors, and mutations in eitherABCC2orABCC3can lead to Cry1Ac-toxin resistance, although this process is not fully understood. In this study, we applied both forward and reverse genetic analyses to demonstrate that high-level Bt-Cry1Ac resistance inPlutella xylostellarequires concurrent mutations in bothPxABCC2andPxABCC3. We identified inactivating mutations in these two genes from a Cry1Ac-resistant strain (Cry1S1000) ofP.xylostellaand conducted genetic linkage analysis, which supported the role thatPxABCC2andPxABCC3were the causal genes of Cry1Ac resistance. We then knocked outPxABCC2andPxABCC3in aP.xylostellasusceptible reference strain (G88) to confirm that high-level Cry1Ac resistance requires mutation ofPxABCC2andPxABCC3, rather than a mutation of either one gene. This finding expands our understanding of complex Bt resistance processes and may be relevant to Bt-Cry1Ac resistance in other lepidopteran insects.The diamondback moth,Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacteriumBacillus thuringiensis(Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal thatP.xylostellarequires combined mutations in bothPxABCC2andPxABCC3to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations ofPxABCC2andPxABCC3that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) ofP.xylostella, with a mutation (R-A2) causing the mis-splicing ofPxABCC2and another mutation (R-A3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed thatR(A2)andR(A3)were tightly linked to Cry1Ac resistance. Introgression ofR(A2)andR(A3)enabled a susceptible strain (G88) ofP.xylostellato obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role ofPxABCC2andPxABCC3in Cry1Ac resistance, frameshift mutations were introduced intoPxABCC2andPxABCC3singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion ofPxABCC2orPxABCC3alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance inP.xylostellaby demonstrating mutations within bothPxABCC2andPxABCC3genes are required for high-level Cry1Ac resistance.