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Virus-mediated transient expression techniques enable genetic modification of Alopecurus myosuroides

View ORCID ProfileMacarena Mellado-Sánchez, View ORCID ProfileFaye McDiarmid, View ORCID ProfileVictor Cardoso, View ORCID ProfileKostya Kanyuka, View ORCID ProfileDana R. MacGregor
doi: https://doi.org/10.1101/2020.01.28.923466
Macarena Mellado-Sánchez
1Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire, AL5 2JQ, UK
2University of Hertfordshire, Department of Biological and Environmental Sciences, School of Life and Medical Sciences, Hatfield, Hertfordshire, AL10 9AB, UK
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Faye McDiarmid
1Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire, AL5 2JQ, UK
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Victor Cardoso
1Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire, AL5 2JQ, UK
3University of Plymouth, School of Biological and Marine Sciences, Portland Square Building, Drake Circus, Plymouth PL4 8AA, UK
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Kostya Kanyuka
1Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire, AL5 2JQ, UK
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Dana R. MacGregor
1Rothamsted Research, Department of Biointeractions and Crop Protection, Harpenden, Hertfordshire, AL5 2JQ, UK
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  • For correspondence: dana.macgregor@rothamsted.ac.uk
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Abstract

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.

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Posted January 29, 2020.
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Virus-mediated transient expression techniques enable genetic modification of Alopecurus myosuroides
Macarena Mellado-Sánchez, Faye McDiarmid, Victor Cardoso, Kostya Kanyuka, Dana R. MacGregor
bioRxiv 2020.01.28.923466; doi: https://doi.org/10.1101/2020.01.28.923466
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Virus-mediated transient expression techniques enable genetic modification of Alopecurus myosuroides
Macarena Mellado-Sánchez, Faye McDiarmid, Victor Cardoso, Kostya Kanyuka, Dana R. MacGregor
bioRxiv 2020.01.28.923466; doi: https://doi.org/10.1101/2020.01.28.923466

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