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 HPPD^Y342H (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). AtHPPD^Y342H mutants exhibited similar phenotype and showed no significant differences in plant height and seed yield with the WT plants (Figure S4).

Figure 1

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Target-site-mutations improve plant resistance to HPPD-inhibitors. The AtHPPD^Y342H mutation significantly increases tolerance to mesotrione (MST) and isoxaflutole (IFT) during germination (a) and seedling (b). Bar equals 3 cm (a) or 7.5 cm (b). (c) Survival rate of the wild type (WT) Col-0 and AtHPPD^Y342H mutants after being sprayed with herbicides at the seedling stage. Phenotype of the soybean WT and GmHPPD^Y388H mutants after being sprayed with MST (d) or IFT (e). Bar equals 15 cm. (f) Seed amount and yield from soybean WT and GmHPPD^Y388H mutants grown in greenhouse without herbicide treatment. ns, no significant difference. (g) Field test for soybean WT and OE-GmHPPD^Y388H mutants with MST and IFT and the resulted seed amount and yield (h). * indicates P < 0.05 in the two-tailed Student’s t-test. Bar equals 20 cm. (i) Rice seeds germinated on the medium containing indicated HPPD-inhibitors. TMT (tembotrione), NIP (Nipponbare), XS134 (Xiushui134). Bar equals 3 cm. (j) Phenotype of rice WT plants, OsHPPD^N338D and OsHPPD^P336L mutants after being sprayed with the indicated herbicides. Bar equals 5 cm.

We transformed the AtHPPD^WT and AtHPPD^Y342H genes under the native promoter into Arabidopsis. As expected, the AtHPPD^Y342H transgenic lines exhibited higher mesotrione tolerance than the AtHPPD^WT transgenic plants (Figure S3a), even though the transgenic AtHPPD^Y342H had equal or lower expression levels compared with AtHPPD^WT (Figure S3b). Together, these results showed that the AtHPPD^Y342H 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 GmHPPD^Y388H lines by base editing, and the resulting homozygous and T-DNA-free GmHPPD^Y388H 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 GmHPPD^Y388H mutants were tolerant up to 58.9 μM of mesotrione (Figure 1d). WT and GmHPPD^Y388H plants were seriously affected by 2.8 μM and 55.7 μM of isoxaflutole, respectively (Figure 1e). The GmHPPD^Y388H 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 GmHPPD^Y388H mutants and WT plants (Figure 1f), indicating that there was no fitness cost for the herbicide tolerance.

We then overexpressed the GmHPPD^Y388H 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).