Engineering Next-Generation Herbicide-Tolerance Alleles Through Protein Design and Gene-Expression Tuning

Weeds pose a major threat to global crop productivity, while decades of reliance on herbicides targeting a limited number of enzymes have accelerated the widespread emergence of herbicide-resistant weeds. Developing crops with tolerance to additional herbicides is therefore crucial for expanding weed-management options and improving agricultural productivity. However, conventional genome-editing strategies often focus on modifying either a gene’s protein-coding sequence or its regulatory elements independently, making it challenging to enhance herbicide tolerance while preserving the gene’s essential biological functions.

Now, our team led by Prof. GAO Caixia and Prof. CHEN Yuhang at the Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), has developed an innovative “dual-layer” allele-engineering strategy to address this challenge. By integrating structure-guided protein design with precise cis-regulatory fine-tuning, we successfully engineered superior herbicide-tolerance alleles in rice and wheat, offering a promising approach to developing herbicide-tolerant crops. The findings were published in Nature Plants on October 12, 2026

We focused on rice 4-hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme involved in tyrosine metabolism and a key target of β-triketone herbicides. By determining the crystal structure of HPPD in complex with the herbicide mesotrione, the team gained insights into the molecular basis of herbicide recognition. Guided by the structural features of the herbicide-binding pocket, we designed and screened amino acid substitutions to reduce herbicide sensitivity. Although several mutations enhanced herbicide resistance, they simultaneously compromised the enzyme’s native activity and adversely affected plant growth, highlighting a fundamental trade-off between herbicide tolerance and essential biological functions.

To overcome this trade-off, we identified a 22-bp expression-modulating element (EME) and precisely introduced it into the regulatory region of the endogenous HPPD gene using prime editing. We observed that combining the EME with the coding-sequence variants enhanced herbicide tolerance synergistically. Notably, some engineered rice lines tolerated mesotrione at four times the recommended field application rate and exhibited resistance to other HPPD-inhibiting herbicides. Field trials further revealed that the balance between herbicide tolerance and agronomic performance was strongly influenced by allelic configuration. While certain combinations negatively affected seed setting and grain yield, retaining one wild-type allele maintained high herbicide tolerance while minimizing adverse effects on agronomic traits.

We further extended this strategy to allohexaploid wheat by editing the coding and regulatory regions of the endogenous HPPD gene to generate lines carrying both the designed protein variant and the EME. These engineered wheat lines exhibited increased HPPD expression and significantly enhanced tolerance to mesotrione compared with wild-type plants, demonstrating the applicability of this approach to crops with complex polyploid genomes.

By jointly optimizing protein function and gene expression, this dual-layer allele-engineering strategy expands the possibilities for developing beneficial alleles that are difficult to achieve through natural variation, conventional mutagenesis, or single-layer genome editing. Importantly, the compact EME enables precise tuning of endogenous gene expression without introducing foreign gene cassettes. As its underlying sequence motifs are naturally widespread in crop genomes, this approach offers a promising and potentially broadly applicable strategy for crop improvement.

More broadly, our work provides a general framework for engineering dosage-sensitive agronomic traits, in which a gain in one function must be balanced against an essential biological role. It offers an effective strategy and technical route for the precise improvement of complex crop traits and for sustainable weed management.

A dual-layer strategy for engineering elite herbicide-tolerance alleles