Generation of imidazolinone herbicide resistant trait in Arabidopsis.

Huirong Dong,Linjian Jiang,Yuexia Zhang,Yuge Yuan,Delin Wang,Zhijing Bai,Hanwen Ni,Wei Yang,Shri Mohan Jain

doi:10.1371/journal.pone.0233503

Abstract

Recently-emerged base editing technologies could create single base mutations at precise genomic positions without generation DNA double strand breaks. Herbicide resistant mutations have been successfully introduced to different plant species, including Arabidopsis, watermelon, wheat, potato and tomato via C to T (or G to A on the complementary strand) base editors (CBE) at the P197 position of endogenous acetolactate synthase (ALS) genes. Additionally, G to A conversion to another conserved amino acid S653 on ALS gene could confer tolerance to imidazolinone herbicides. However, no such mutation was successfully generated via CBE, likely due to the target C base is outside of the classic base editing window. Since CBE driven by egg cell (EC) specific promoter would re-edit the wild type alleles in egg cells and early embryos, we hypothesized the diversity of base editing outcomes could be largely increased at later generations to allow selection of desired herbicide resistant mutants. To test this hypothesis, we aimed to introduce C to T conversion to the complement strand of S653 codon at ALS gene, hosting a C at the 10th position within the 20-nt spacer sequence outside of the classic base editing window. While we did not detect base-edited T1 plants, efficient and diverse base edits emerged at later generations. Herbicide resistant mutants with different editing outcomes were recovered when T3 and T4 seeds were subject to herbicide selection. As expected, most herbicide resistant plants contained S653N mutation as a result of G10 to A10. Our results showed that CBE could create imidazolinone herbicide resistant trait in Arabidopsis and be potentially applied to crops to facilitate weed control.

Highlights

CRISPR/Cas9 could efficiently induce DNA double strand breaks (DSB), followed by either non-homology end joining (NHEJ) or homology directed repair (HDR) in plant cells [1]
Analysis of known herbicide resistant mutations in plants showed that the herbicide target acetolactate synthase (ALS), known as acetohydroxy acid synthase (AHAS), harbors two potential herbicide resistant mutations as a result of C to T conversion [5]
Arabidopsis plants were transformed by floral dip method and transgenic plants were selected on MS medium supplemented with hygromycin at 25 mg L-1

Summary

Introduction

CRISPR/Cas could efficiently induce DNA double strand breaks (DSB), followed by either non-homology end joining (NHEJ) or homology directed repair (HDR) in plant cells [1]. In plant and mammalian cells, the predominating NHEJ pathway efficiently yielded insertion/ deletion (indels) mutations that largely resulted in loss-of-function outcomes [2]. Analysis of known herbicide resistant mutations in plants showed that the herbicide target acetolactate synthase (ALS), known as acetohydroxy acid synthase (AHAS), harbors two potential herbicide resistant mutations as a result of C to T (or G to A on the complementary strand) conversion [5]. The S653 codon of wheat ALS gene was subject to CBE, resulting in only G654 and G655 edits [8]. The S653N mutation as a result of C to T transition on the complimentary strand was not achieved via CBE

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