Identifying resistant mutations in the herbicide target site of the plant 4‐hydroxyphenylpyruvate dioxygenase

Weed species have increasingly emerged with resistance against previously effective herbicides, such as glyphosate and inhibitors of acetyl coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) (Heap, 2024). Owing to its novel mode of action, 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitors are effective in controlling herbicide-resistant weeds and recently attracted much attention. Resistance to HPPD-inhibitors has been slow to evolve in weeds, and only a few cases of resistant events have been reported and most of these are associated with enhanced herbicide metabolism (Heap, 2024; Lu et al., 2023). Since resistant sites in the entire target gene are largely unknown, we in vivo mutagenized the HPPD gene in Arabidopsis and rice using base editing libraries to uncover potential target-site resistant mutations. Arabidopsis are highly sensitive to mesotrione. After large-scale transformation of base editing pools (Figure S1), we found one T1 seedling growing normally in medium containing 100 nM mesotrione (Figure S2a). Genotyping showed that a heterozygous T-to-C substitution occurred, causing the Y342H mutation in the HPPD protein sequence (Figure S2b, c). Homozygous progenies of HPPDY342H (Figure S2d) were tolerant up to 200 nM mesotrione and 50 nM isoxaflutole in medium (Figures 1a and S3a) and had a much higher survival rate than wild type (WT) when sprayed with ≥1 μM mesotrione or isoxaflutole in pots (Figure 1b, c). AtHPPDY342H mutants exhibited similar phenotype and showed no significant differences in plant height and seed yield with the WT plants (Figure S4). We transformed the AtHPPDWT and AtHPPDY342H genes under the native promoter into Arabidopsis. As expected, the AtHPPDY342H transgenic lines exhibited higher mesotrione tolerance than the AtHPPDWT transgenic plants (Figure S3a), even though the transgenic AtHPPDY342H had equal or lower expression levels compared with AtHPPDWT (Figure S3b). Together, these results showed that the AtHPPDY342H mutation significantly increases the target-site resistance to HPPD-inhibitors. We next determined whether this mutation also increased tolerance in soybean, a crop highly sensitive to HPPD-inhibitors. Alignment of protein sequences between Arabidopsis and soybean showed that the Y388 of GmHPPD corresponds to the Y342 of AtHPPD. We generated the GmHPPDY388H lines by base editing, and the resulting homozygous and T-DNA-free GmHPPDY388H offsprings in the T3 generation were treated with mesotrione and isoxaflutole. Our results showed that WT plants were suppressed by 2.9 μM mesotrione, whereas the GmHPPDY388H mutants were tolerant up to 58.9 μM of mesotrione (Figure 1d). WT and GmHPPDY388H plants were seriously affected by 2.8 μM and 55.7 μM of isoxaflutole, respectively (Figure 1e). The GmHPPDY388H mutants were estimated to increase 20-fold resistance to HPPD-inhibitors than WT plants. Importantly, consistent with the results obtained from Arabidopsis, the seed amount and yield did not significantly differ between GmHPPDY388H mutants and WT plants (Figure 1f), indicating that there was no fitness cost for the herbicide tolerance. We then overexpressed the GmHPPDY388H in soybean to further improve herbicide resistance (Figure S5). Owing to the combined effect of increased expression level and target-site mutation, transgenic offsprings showed a 100% survival rate albeit with ~10% yield reduction under 590 μM mesotrione or 278 μM isoxaflutole treatment (Figure 1g), which are in the range of concentrations required for weed control in the field (300–600 μM). Unlike Arabidopsis and soybean, Japonica rice is resistant to triketones due to an endogenous HIS1 gene that detoxifies these herbicides (Maeda et al., 2019). To further increase their resistance by target-site mutations, we pool-edited the HPPD gene in Japonica rice using base editors. Rice exhibits high sensitivity to HPPD-inhibitors at seed germination stage, so we screened for resistant mutants using T1 seeds. This strategy also enabled us to simultaneously test different HPPD-inhibitors (Figure S6). Hundreds of amino acid mutations were identified from randomly examined transgenic lines (Figure S7; Table S1). Although most of these mutants remained sensitive, several mutants exhibited increased resistance to one or more of the tested herbicides (Data S1). The N338D mutation resulted in a slight tolerance to mesotrione, tembotrione and isoxaflutole at the germination stage, but only isoxaflutole-resistance was confirmed at the seedling stage (Figure 1i, j). The P336L mutation increased resistance to tembotrione but not mesotrione or isoxaflutole (Figure 1i, j). Both the N338D and P336L mutants showed decreased plant height and/or lower seed setting rate (Figure S8), indicating that there were fitness cost for the herbicide tolerance. The Y339H mutation, which corresponds to the AtHPPD-Y342H, did not improve tolerance to any of the tested HPPD-inhibitors (Data S1). Since the HIS1 gene makes a major contribution to the resistance, it is not surprising that mutations in OsHPPD only slightly increase the overall tolerance to HPPD-inhibitors. Very recently, the HPPD coding sequences of cotton and Arabidopsis were also evolved in vitro or in vivo (Qian and Shi, 2024; Wang et al., 2024). Most of the resistant mutations are located within or near the helix gate, which may alter the binding site conformation and affect herbicide accessibility. Nevertheless, the increased resistance endowed by these mutations alone might not be sufficient for application in the field, which might explain why almost no target-site resistant weeds have been reported thus far. With the long-term application of HPPD herbicides, emerging resistant weeds likely have enhanced metabolism of the specific types of herbicides, which implies that rotating use of different types of HPPD herbicides may help to delay the emergence of resistant weeds. Recommendable strategies for improving crop tolerance include combined the target-site mutations with a higher background-tolerance HPPD gene such as that of maize (Siehl et al., 2014), enhanced expression by ectopically expressing HPPD gene in plastids (Dufourmantel et al., 2007) or directly knocking-up the endogenous HPPD gene (Lu et al., 2021), and introduced the HPPD-inhibitor metabolism gene HIS1 (Maeda et al., 2019). Variations in 3’-UTR of the OsHPPD, which may affect the regulation of mRNA stability or protein translation, have also been reported to improve resistance (Wu et al., 2023). This work was supported by National Natural Science Foundation of China (32188102 to J.-K.Z., 31901046 and 32271524 to M.W.) and Nanfan special project, CAAS (ZDXM2314). The authors declare no competing interests. J.-K.Z., M.W., Y.M., and Y.Z. conceived of and designed the research. Y.M., Y.Z., and J.X. prepared the tests for Arabidopsis. M.W., Y.H., Y.W., and H.W. conducted the experiments for rice. M.W., H.X., L.X., and X.W. tested the mutations in soybean. G.Z. and V.V. provided helpful suggestions. J.-K.Z., M.W. and Y.M. wrote the manuscript. Figure S1 Procedure for obtaining HPPD-resistant mutations in Arabidopsis via base editing. Figure S2 Identification of the HPPDY342H resistant mutation in Arabidopsis. Figure S3 AtHPPDY342H transgenic lines showed much higher resistance to mesotrione (MST) than AtHPPDWT transgenic lines. Figure S4 The AtHPPDY342H-edited mutants showed similar growth and development phenotype with wild type plants. Figure S5 GmHPPDY388H -overexpression line showed resistance to high concentration of mesotrione (MST). Figure S6 Procedure of base editing-mediated mutagenesis in rice HPPD for herbicide-resistant mutants. Figure S7 Identified HPPD mutations from randomly selected rice transgenic lines and their distribution across the first exon (a) and the second exon (b). Figure S8 Plant height and seed setting rate of rice wild type, HPPDP336L, and HPPDN338D mutant plants. Table S1 Quality of the sgRNA libraries and mutagenesis efficiency in editing the rice HPPD gene. Table S2 Primers used in this study. Supplemental Sequences. Data S1 Herbicides-resistance of some identified rice HPPD mutants. Data S2 Guide Oligos for Arabidopsis Library. Data S3 Guide Oligos for Rice Library. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.