Virus-mediated transient expression techniques enable genetic modification of Alopecurus myosuroides
Virus-mediated transient expression techniques enable genetic modification of Alopecurus myosuroides
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DOI：
10.1101/2020.01.28.923466
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发表时间：
2020-01
期刊：
影响因子：
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通讯作者：
Macarena Mellado-Sánchez;F. McDiarmid;V. Cardoso;K. Kanyuka;D. MacGregor
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文献类型：
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作者：
Macarena Mellado-Sánchez;F. McDiarmid;V. Cardoso;K. Kanyuka;D. MacGregor
尽管杂草生态学已经取得了相当大的进展，但杂草分子生物学一直受到无法遗传操纵杂草的阻碍。遗传操作对于证明基因型和表型之间的因果关系至关重要。在这里，我们证明，病毒介导的瞬时表达技术开发的其他单子叶植物可以用于黑草（大穗看麦娘）的损失和获得的功能研究。我们不仅使用病毒诱导的基因沉默（VIGS）来产生表现出降低的八氢番茄红素脱饱和酶表达和病毒介导的过表达（VOX）来驱动绿色荧光蛋白的黑草，我们证明这些技术适用于测试与黑草中除草剂抗性相关的假设。我们使用VIGS证明，AmGSTF1是必要的抗性生物型Peldon生存fenoxaprop应用和显示异源表达的双丙氨膦抗性基因与VOX是足够的，赋予抗性，否则致死剂量的草铵膦。对于英国和西欧的冬季谷物农民来说，黑草是最有问题的杂草，因为它迅速进化出适应性，使其能够有效地避免目前的综合杂草管理做法。黑种草还降低产量，因此直接威胁粮食安全和生产力。因此，需要新的破坏性技术，减轻抗性进化，并能够更好地控制这种有害杂草。这些病毒介导的方案为我们在受控实验室条件下改变感兴趣基因的能力提供了一个步骤，从而获得了对黑草如何在农业环境中生存的分子水平的理解。病毒介导的瞬时表达技术在黑草中产生功能丧失和获得突变，并显示特定基因型与除草剂抗性可测量变化之间的因果关系。
Even though considerable progress has been made in weed ecology, weed molecular biology has been hindered by an inability to genetically manipulate weeds. Genetic manipulation is essential to demonstrate a causative relationship between genotype and phenotype. Herein we demonstrate that virus-mediated transient expression techniques developed for other monocots can be used in black-grass (Alopecurus myosuroides) for loss- and gain-of-function studies. We not only use virus induced gene silencing (VIGS) to create the black-grass exhibiting reduced PHYTOENE DESATURASE expression and virus-mediated overexpression (VOX) to drive GREEN FLUORESCENT PROTEIN, we demonstrate these techniques are applicable to testing hypotheses related to herbicide resistance in black-grass. We use VIGS to demonstrate that AmGSTF1 is necessary for the resistant biotype Peldon to survive fenoxaprop application and show the heterologous expression of the bialaphos resistance gene with VOX is sufficient to confer resistance to an otherwise lethal dose of glufosinate. Black-grass is the most problematic weed for winter-cereal farmers in the UK and Western Europe as it has rapidly evolved adaptions that allow it to effectively avoid current integrated weed management practices. Black-grass also reduces yields and therefore directly threatens food security and productivity. Novel disruptive technologies which mitigate resistance evolution and enable better control over this pernicious weed are therefore required. These virus-mediated protocols offer a step change in our ability to alter genes of interest under controlled laboratory conditions and therefore to gain a molecular-level understanding of how black-grass can survive in the agri-environment. One Sentence Summary Virus-mediated transient expression techniques create loss- and gain-of-function mutations in black-grass and show causation between specific genotypes and measurable changes in herbicide resistance.