A rice gene that confers broad-spectrum resistance to β-triketone herbicides

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Science  26 Jul 2019:
Vol. 365, Issue 6451, pp. 393-396
DOI: 10.1126/science.aax0379

A double-edged rice paddy herbicide

A useful herbicide kills weeds but spares the crop of interest. For many rice paddies, the herbicide benzobicyclon (BBC) serves this purpose. But some rice strains are susceptible to BBC, which diminishes its value in weed control. Maeda et al. uncovered the genetic cause controlling the response to the herbicide: resistant rice cultivars have an oxidase that detoxifies BBC herbicides. Susceptible rice varieties carried genetic mutations that disabled the oxidase. The gene HPPD INHIBITOR SENSITIVE 1, which encodes the oxidase, may be useful for developing BBC-resistant crops.

Science, this issue p. 393


The genetic variation of rice cultivars provides a resource for further varietal improvement through breeding. Some rice varieties are sensitive to benzobicyclon (BBC), a β-triketone herbicide that inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD). Here we identify a rice gene, HIS1 (HPPD INHIBITOR SENSITIVE 1), that confers resistance to BBC and other β-triketone herbicides. We show that HIS1 encodes an Fe(II)/2-oxoglutarate–dependent oxygenase that detoxifies β-triketone herbicides by catalyzing their hydroxylation. Genealogy analysis revealed that BBC-sensitive rice variants inherited a dysfunctional his1 allele from an indica rice variety. Forced expression of HIS1 in Arabidopsis conferred resistance not only to BBC but also to four additional β-triketone herbicides. HIS1 may prove useful for breeding herbicide-resistant crops.

Rice (Oryza sativa L.) is a staple food for >3.5 billion people (1). Genetic variation revealed by characterization of genomes of the genus Oryza, including domesticated and wild species, has allowed the identification of genes useful for crop breeding (24). Efficient crop production in large-scale farming depends on weed control with herbicides, but long-term use of such agents can lead to the appearance of resistant weeds (510). New combinations of herbicides and herbicide resistance genes are therefore needed for crop breeding (1114).

β-Triketone herbicides (bTHs) are 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and are applied widely in agriculture (8). The bTH benzobicyclon (BBC) (Fig. 1A), developed for weed control in rice paddy fields (15), shows efficacy against paddy weeds resistant to other types of herbicide, including sulfonylureas. However, some high-yield rice varieties are susceptible to BBC (Fig. 1, B and C), with failure to identify the gene responsible for BBC sensitivity having put future rice breeding programs at risk. We therefore set out to identify the rice gene that determines BBC resistance or sensitivity.

Fig. 1 Identification and characterization of a BBC resistance gene from japonica rice.

(A) Structures of BBC and its hydrolysate, BBC-OH. (B) The cultivars Koshihikari, Momiroman, and Habataki were grown in a paddy field treated with BBC (300 g of active ingredient/ha) at 7 and 21 days after transplantation. Examination of the plants at 40 days after transplantation revealed that Koshihikari is resistant to BBC, whereas Momiroman and Habataki are sensitive. (C) Concentration-dependent effects of BBC on Koshihikari (HIS1) and Momiroman (his1) grown in a controlled environment. (D) Summary of map-based cloning of HIS1 from japonica rice. (E) Concentration-dependent effects of BBC on the cultivar Nipponbare (NB, HIS1) and the his1 homozygous mutant lines NG6511 and NF8046 grown in a controlled environment. (F) Structures of four bTHs. (G) Effects of bTHs (SLT, 0.14 μM; MST, 0.13 μM; TFT, 3.07 μM) on Nipponbare, NG6511 (NG), and NF8046 (NF) grown in a controlled environment.

We first performed map-based cloning. Analysis of quantitative trait loci (QTLs) with a BC1F2 population and chromosome segment substitution lines (16) derived from BBC-sensitive and BBC-resistant rice varieties identified a major QTL for BBC susceptibility on chromosome 2 (Fig. 1D). The BBC sensitivity determined by this QTL behaved as a single recessive trait, and we designated the corresponding wild-type gene HIS1 (HPPD INHIBITOR SENSITIVE 1). The HIS1 locus was narrowed down to a 116.4-kb genomic interval that contained 11 candidate genes (table S1), of which Os02g0280700 was predicted to encode a 351–amino acid protein with a deduced sequence that includes motifs conserved in a family of Fe(II)/2-oxoglutarate (2OG)–dependent oxygenases (fig. S1). Nucleotide sequence analysis revealed that BBC-sensitive cultivars harbor a nonsense mutation caused by a 28–base pair (bp) deletion in exon IV of this gene (Fig. 1D and fig. S1). Two Os02g0280700 mutant lines, NG6511 and NF8046, which harbor an insertion of the retrotransposon Tos17 (17) within exons I and V, respectively (Fig. 1D), manifested a bleached phenotype under BBC treatment (Fig. 1E). Analysis of self-pollinated offspring derived from heterozygous parents of these lines confirmed that their genotypes were 100% linked to BBC sensitivity or resistance (table S2). The mutants were sensitive to sulcotrione (SLT) and mesotrione (MST) (Fig. 1, F and G), indicating that Os02g0280700 functions as a resistance gene for other bTHs as well as BBC. No differences in other agronomic traits were apparent between the wild type (Nipponbare) and either NG6511 or NF8046. We therefore concluded that Os02g0280700 is HIS1 and that HIS1 is dispensable for normal growth and development in rice. Analysis of breeding history by polymerase chain reaction (PCR)–based genotyping suggested that the 28-bp deletion allele (his1) of HIS1 originated in Peta, an indica cultivar from Indonesia (fig. S1) (18). The deletion was not found in most japonica varieties but is present in Tadukan and IR64, which are parent cultivars of modern indica varieties (fig. S1 and table S3).

In a previous survey of plant Fe(II)/2OG-dependent oxygenases (19), HIS1 is categorized in a clade that includes proteins of unknown function. Our phylogenetic analysis revealed that HIS1-like (HSL) genes are highly conserved among poaceous species (Fig. 2A and fig. S2). The rice (Nipponbare) genome harbors multiple HSL genes, five (OsHSL1/2/4/5/6) of which are present in a gene cluster on chromosome 6. Among the predicted OsHSL proteins, Os06g0176700 (designated OsHSL1) shows the closest similarity (87% sequence identity) to HIS1 (fig. S2). Results from three-dimensional homology modeling suggest that HIS1 and OsHSL1 share a double-stranded β-helix fold, which is a typical structural signature of Fe(II)/2OG-dependent oxygenases (20) (fig. S3). Nongrass monocot species also share a group of HSL genes independent of grass HSL genes, with dicot species also sharing HSL-related genes, suggestive of common functions for HSL genes in the plant kingdom (Fig. 2B).

Fig. 2 Phylogenetic analysis of HIS1 and its related proteins.

Phylogenetic trees are shown for HIS1 and HSL proteins in grass species (A) and for HIS1, HSL, and related 2OG-dependent oxygenase (2OGD) proteins in the plant kingdom (B). The trees were generated with the neighbor-joining method together with the bootstrap test. Os, Oryza sativa; Hv, Hordeum vulgare; Ta, Triticum aestivum; Zm, Zea mays; Sb, Sorghum bicolor; At, Arabidopsis thaliana.

The Rice Expression Profile Database (RiceXPro) contains data on the expression specificity of HIS1 and OsHSL1 (fig. S4 and table S4). To confirm the expression patterns of these genes in rice, we performed reverse transcription (RT)–PCR analysis. HIS1, as well as each of the OsHSL genes, was found to be expressed in plants at different levels and with different tissue specificity (fig. S4 and table S4). HIS1 was preferentially expressed in leaves of young plants, whereas OsHSL1 was expressed in leaves at a high level and in roots at a lower level throughout development.

We next subjected a rice his1 variety (Yamadawara) to genetic transformation with HIS1 or OsHSL1 cDNA (fig. S5). Expression of HIS1 conferred resistance to BBC, confirming its identification as the gene responsible for BBC resistance (Fig. 3A). Transformed lines expressing HIS1 also manifested increased resistance to other bTHs—including SLT, MST, tembotrione (TMT), and tefuryltrione (TFT) (Fig. 1F)—compared with Nipponbare (Fig. 3A). Sensitivity to non–bTH-type HPPD inhibitors, such as isoxaflutole and pyrazolate, was not affected in the transgenic plants (table S5). Although transformed lines expressing OsHSL1 did not manifest BBC resistance, they showed increased resistance to TFT (fig. S5). The bTH-sensitive model plant Arabidopsis thaliana became resistant to BBC, SLT, MST, TFT, and TMT after transformation with HIS1 (Fig. 3B). These results thus suggested the possible application of HIS1 to the breeding of crops resistant to multiple bTHs. Arabidopsis lines expressing OsHSL1 showed resistance only to TFT among bTHs (Fig. 3B). HIS1 mutant rice lines show resistance to TFT but are sensitive to MST and SLT as well as BBC (Figs. 1G and 3A), suggesting that OsHSL1 may contribute to natural TFT resistance in rice together with HIS1.

Fig. 3 Herbicide susceptibility of transgenic plants.

(A) Herbicide susceptibility of Yamadawara transformed with HIS1 cDNA. T3 seeds of two different transgenic lines (#5, #6) were grown on Murashige-Skoog (MS) solid medium containing the indicated herbicide concentrations. P35S, 35S promoter of cauliflower mosaic virus. (B) Herbicide susceptibility of A. thaliana columbia (Col) transformed with HIS1 or OsHSL1. Homozygous plant seeds were germinated and grown on MS solid medium in the absence or presence of BBC (0.05 μM), SLT (0.05 μM), MST (0.05 μM), TFT (0.02 μM), or TMT (0.02 μM). Kanamycin (Kan) was used for positive selection of transformed plants.

To clarify the function of HIS1 in BBC metabolism, we investigated the turnover of BBC and its derivatives in rice. BBC is a prodrug, with its hydrolysate, BBC-OH (Fig. 1A), being incorporated, serving as an HPPD inhibitor, and mediating plant bleaching (15). Analysis of the BBC-sensitive cultivar Yamadawara revealed that BBC-OH was absorbed into roots and transferred to foliar tissue (table S6). Although it was detected in roots, BBC-OH was not detected in leaf tissue of HIS1-transformed Yamadawara (table S6), suggesting that HIS1 functions as an enzyme for BBC-OH degradation in the plant body.

We next prepared recombinant HIS1 with a cell-free translation system (21) to further explore the role of this protein in herbicide inactivation (fig. S6). High-performance liquid chromatography (HPLC) revealed that HIS1 catalyzes BBC-OH metabolism in the presence of Fe2+ and 2OG (Fig. 4A). Assay of HPPD activity in the presence of BBC-OH or the isolated BBC-OH metabolite showed that the latter had lost the ability to inhibit HPPD (Fig. 4B). Liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS) revealed that the molecular weight of the BBC-OH metabolite generated by HIS1 exceeded that of BBC-OH by 16 (Fig. 4C), indicating incorporation of a single oxygen atom. To identify the site of the hydroxylation, we synthesized 3-acetyl-bicyclo[3.2.1]?octane-?2,?4-?dione (3-acetyl BOD) (Fig. 4D) as a mimic for a portion of the BBC-OH structure. HPLC and LC-ESI-MS analyses showed that 3-acetyl-BOD was converted to a hydroxylated derivative by HIS1 (Fig. 4D and table S7). Nuclear magnetic resonance (NMR) revealed that this derivative possesses a hydroxyl group at the C8 position and was therefore identified as 8-OH-3-acetyl-BOD (Fig. 4D and table S7). The BBC-OH metabolite yielded two peaks (peaks 1 and 2) in Fig. 4C, and 8-OH-3-acetyl-BOD appeared as partially overlapping peaks in Fig. 4D. These results suggested that at least two isomers are generated as a result of the formation of the hydroxyl group in BBC-OH and at the C8 position of 3-acetyl-BOD. The mass/charge (m/z) ratios for the cleavage fragment of BBC-OH (165, peak 3) and for peaks 1 and 2 (181 for each) also indicate that HIS1 catalyzes hydroxylation at the corresponding position of BBC-OH (Fig. 4C and fig. S7). We thus conclude that BBC-OH undergoes hydroxylation at the corresponding position in a reaction catalyzed by HIS1 as an Fe(II)/2OG-dependent oxygenase (Fig. 4E). HPLC showed that HIS1 also modifies TFT, SLT, and MST as well as BBC-OH (fig. S8 and table S8), consistent with the results of herbicide susceptibility tests for HIS1-expressing plants. LC-ESI-MS analysis revealed that the compounds generated by HIS1 from these bTHs all had a molecular weight greater than that of the parent molecule by 16 (fig. S8), suggesting that HIS1 catalyzes the hydroxylation of TFT, SLT, and MST as it does that of BBC-OH.

Fig. 4 Enzyme reaction catalyzed by HIS1.

(A) HPLC analysis of reaction mixtures after incubation of recombinant HIS1 with BBC-OH in the presence of Fe2+ and in the absence (upper trace) or presence (lower trace) of 2OG. In the presence of 2OG, BBC-OH is converted to its metabolite by the catalysis of HIS1. mAU, milli–absorbance unit. (B) Activity of carrot HPPD measured in the presence of the indicated concentrations (horizontal axis) of BBC-OH or of the isolated BBC-OH metabolite identified in (A). Vertical axis shows relative rate (%) of inhibition of HPPD activity by the compounds. Data are means of triplicates from a representative experiment. (C) HPLC separation of the BBC-OH metabolite generated as in (A) for analysis by LC-ESI-MS. Peak 3 corresponds to BBC-OH, and peaks 1 and 2, which were not separated by the HPLC condition in (A), correspond to the BBC-OH metabolite. Determined m/z ratios are shown in the inset. MW, molecular weight. (D) HPLC profiles for 3-acetyl-BOD and its metabolite, 8-OH-3-acetyl-BOD, generated by HIS1. The molecular weight of each compound determined by LC-ESI-MS analysis (fig. S7) is shown in red. (E) Reaction scheme for the metabolism of BBC-OH by HIS1 based on the results of LC-ESI-MS (fig. S7) and NMR (table S7) analyses of the BBC-OH metabolite. [(A) to (D)] Details are described in materials and methods.

We have isolated and characterized a herbicide resistance gene, HIS1, from japonica rice and demonstrated that the encoded enzyme catalyzes the hydroxylation of several bTHs. The enzyme requires Fe2+ and 2OG for catalysis and therefore belongs to the Fe(II)/2OG-dependent oxygenase family. This function of the HIS1 protein was confirmed by gene transformation in Arabidopsis and in a rice his1 mutant. Given that HIS1 functions as a single gene to detoxify multiple bTHs, its application to molecular crop breeding will support multiple options for herbicide choice. We also identified a second herbicide resistance gene, OsHSL1, in rice and showed that forced OsHSL1 expression renders Arabidopsis resistant to TFT. The homology modeling (fig. S3) narrowed down the number of different amino acids in the putative substrate pockets of HIS1 and OsHSL1 to five, suggesting that some of, or a coordination of, these amino acids determines substrate selectivity. This could allow improvements in OsHSL1 function by targeting specific residues for mutation. Given that the spread of weeds resistant to HPPD inhibitors remains limited compared with that of weeds resistant to other herbicides, such as 5-enolpyruvylshikimate-3-phosphate synthase inhibitors and acetolactate synthase inhibitors (9), application of bTHs together with HIS1 or OsHSL1 will be practical for weed control in crop fields.

Supplementary Materials

Materials and Methods

Figs. S1 to S8

Tables S1 to S9

References (2234)

References and Notes

Acknowledgments: We thank I. Ando and Y. Sunohara for rice cultivars; A. Izumi and T. Fujishiro for help with homology modeling; T. Kotake for help with qPCR; and T. Akiyama and H. Masuda for assistance with experiments. Funding: This research was supported by a grant from the Bio-oriented Technology Research Advancement Institution, NARO (Research Program on Development of Innovative Technology, 29009B), and by a grant from NARO (Precedence and Trial Research Project of NARO). Author contributions: Y. Tozawa and H.Y. designed research. H.K. and M.O. arranged the research consortium. H.M., K.M., S.T., S.H., M.K., M.K.-K., Y. Taniguchi, M.O., H.K., H.Y., and Y. Tozawa contributed map-based cloning and transgenic analysis. H.M. and M.K. performed genealogy analysis. H.Y. performed expression, genomic, and phylogenetic analyses. H.M., K.M., N.S., A.Y., M.R.K., K.S., and H.K. analyzed herbicide resistance and evaluated agronomic traits of mutants. A.Y., K.S., and S.H. contributed metabolite analysis. N.S., S.T., S.S., and Y. Tozawa performed protein analysis. Y. Tozawa and H.Y. wrote the paper. All authors discussed the results and approved the manuscript. Competing interests: The authors declare no competing interests. Data and materials availability: All data are available in the manuscript or the supplementary materials. All DNA constructs and plant strains are available from the corresponding author.
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