Location: Grape Genetics Research Unit (GGRU)Title: Amino acid substitutions in grapevine (Vitis vinifera) acetolactate synthase conferring herbicide resistance
Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2023
Publication Date: 5/11/2023
Citation: Wuddineh, W., Xu, X., Zhong, G. 2023. Amino acid substitutions in grapevine (Vitis vinifera) acetolactate synthase conferring herbicide resistance. Plant Cell Tissue and Organ Culture. https://doi.org/10.1007/s11240-023-02512-8.
Interpretive Summary: Herbicide resistance is an important agronomic trait for many crops. One of the key target plant genes for herbicide resistance is the acetolactate synthase (ALS) gene, which is well known for its natural mutations endowing resistance to ALS-inhibiting herbicides in various weedy biotypes and other plant species. In addition to its potential of being an important agronomic trait, ALS mutation-derived herbicide resistance has also been explored as a plant selectable marker in transformation. Currently, there is no known report on grapevine ALS mutations conferring resistance to ALS inhibiting herbicides. In this study, we report our work on the evaluation of responses of grapevine embryogenic callus and in vitro meristematic tissue to ALS-inhibiting herbicides, the creation of transgenic vines expressing three VvALS1 single amino acid substitution mutations and the demonstration of these substitution mutations for high resistance to ALS-inhibiting herbicides namely Imzarapyr or chlorsulfuron. Our results confirmed that mutations in the grapevine ALS gene can be effectively employed as a plant selectable marker in transformation. In addition, we report two ALS-inhibiting herbicides of different chemistry, which were determined to be suitable for resistant plant selection in grapevine.
Technical Abstract: Acetolactate synthase genes (ALS) have successfully been modified for providing resistance to ALS-inhibiting herbicides in many plant species. Based on sequence and expression analyses, we confirmed VvALS1 as the best functional ALS candidate in grapevine. To develop an ALS-based herbicide selection system for facilitating grape transformation, we firstly evaluated the responses of Vitis vinifera cv Chardonnay callus and young in vitro shoots of Vitis vinifera cv Thompson Seedless to several representative ALS-inhibiting herbicides and found a typical linear response curves to some of the herbicides, including chlorsulfuron and imazapyr belonging to the sulfonylurea or imidazolinone families, respectively. Secondly, we created constructs containing amino acid substitutions in the domains which are known to be critical to herbicide resistance and generated transgenic plants for 3 amino acid substitutions using Agrobacterium-mediated transformation of meristematic bulk tissues of Thompson Seedless. Finally, we showed that ectopic expression of two amino acid substitutions (P191S and P191T) at the N-terminal region and another (W568L) at C-terminal region in VvALS resulted in high resistance to chlorsulfuron or imazapyr herbicides in transgenic in vitro shoots. Our work highlighted the potential use of VvALS mutations imparting herbicide resistance as a selectable marker in grapevine transformation research and as a means in fostering grapevine improvement via cisgenesis, paving the way for developing a selectable co-editing system to facilitate transgene-free gene-editing.