Generation of 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide-resistant rice (Oryza sativa L.) germplasm based on enhanced DNA shuffling and CRISPR-mediated base editing.

INTRODUCTION: Weeds significantly threaten rice production. While herbicides are the most efficient weed-control method, prolonged use accelerates resistant weed emergence. 4-Hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides offer potential for managing resistant weeds in rice, but the availability of resistant rice mutants is limited. OBJECTIVES: This study aimed to optimize the method of DNA shuffling to conduct directed evolution of the rice HPPD gene, discover novel resistance mutations, and generate herbicide-resistant rice germplasm. METHODS: We developed an efficient DNA shuffling method utilizing flanking sequences and segmented amplification to construct random mutation libraries of rice HPPD and maize (Zea mays L.) HPPD genes. Mutant libraries were screened via chromogenic reaction in Escherichia coli. CRISPR-mediated adenine base editing was adopted to generate rice mutants, and hydroponic assays were conducted to evaluate herbicide resistance. The crystal structure of rice HPPD was determined by X-ray crystallography, while mutant structures and ligand docking were simulated with AlphaFold3. RESULTS: Flanking sequences enhanced single-stranded DNA amplification efficiency, and segmented amplification reduced nonsense mutations. A resistant mutant, OsHPPD-mHelix, was obtained. Enzyme kinetics analysis demonstrated that its Ki for mesotrione was increased approximately 2.5-fold compared to that of wild-type OsHPPD, while its enzyme activity was not significantly affected. Using the mutation sites contained in OsHPPD-mHelix as hotspots, genome-edited rice was further generated. It was found that the mesotrione resistance of rice carrying the E423G mutation was approximately 2.2-fold higher than that of wild-type rice. Structural and molecular dynamics analyses suggested the E423G mutation promotes a closed terminal α-helix conformation, with free energy landscapes indicating reduced mesotrione binding stability, potentially explaining the resistance mechanism. CONCLUSION: This study provides an efficient DNA shuffling technology and novel herbicide-resistant rice germplasm, enriching the toolbox for rice gene-directed evolution and resistant variety cultivation.