Herbicide-resistant 4-hydroxyphenylpyruvate dioxygenase variants identified via directed evolution.

Herbicides play a crucial role in boosting crop yields, yet the emergence of herbicide-resistant weeds and the susceptibility of crops to herbicides have posed significant challenges to their efficacy. β-Triketone herbicides specifically target the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD), which is essential for plant growth. Remarkably, few resistant weeds have been identified against these herbicides. In this study, we aimed to identify mutations within the cotton HPPD gene that confer resistance to mesotrione, a widely used triketone herbicide. Through the establishment of a high-throughput mutant screening system in Escherichia coli, we identified four single nucleotide changes leading to amino acid substitutions in HPPD, resulting in mesotrione resistance while preserving native enzymatic activity. Various combinations of these mutations displayed synergistic effects on herbicide resistance. Additionally, the HPPD variants were able to complement the Arabidopsis athppd mutant, indicating their retention of sufficient native activity for plant growth and development. Expression of these cotton HPPD variants in Arabidopsis resulted in heightened herbicide resistance. These findings offer critical insights into the target amino acids of HPPD for gene editing, paving the way for the development of herbicide-resistant cotton in the future.