An amino acid transporter-like protein (OsATL15) facilitates the systematic distribution of thiamethoxam in rice for controlling the brown planthopper.

Characterization and genetic engineering of plant transporters involved in the pesticide uptake and translocation facilitate pesticide relocation to the tissue where the pests feed, thus improving the bioavailability of the agrichemicals. We aimed to identify thiamethoxam (THX) transporters in rice and modify their expression for better brown planthopper (BPH) control with less pesticide application. A yeast library expressing 1385 rice transporters was screened, leading to the identification of an amino acid transporter-like (ATL) gene, namely OsATL15, which facilitates THX uptake in both yeast cells and rice seedlings. In contrast to a decrease in THX content in osatl15 knockout mutants, ectopic expression of OsATL15 under the control of the CaMV 35S promoter or a vascular-bundle-specific promoter gdcsPpro significantly increased THX accumulation in rice plants, thus further enhancing the THX efficacy against BPH. OsATL15 was localized in rice cell membrane and abundant in the root transverse sections, vascular bundles of leaf blade, and stem longitudinal sections, but not in hull and brown rice at filling stages. Our study shows that OsATL15 plays an essential role in THX uptake and its systemic distribution in rice. OsATL15 could be valuable in achieving precise pest control by biotechnology approaches.

Introduction

Agrochemicals applied to enhance crop productivity and protect plants from pests and diseases threaten the health of soil and water, as well as animal and plant communities (Sun et al.,  2012). A potential strategy to reduce chemical input while maintaining optimal crop yield is to improve the bioavailability of the agrochemicals (David, 2016). The bioavailability of agrochemicals is determined by the dosage of the active ingredient applied to the plant (aerial parts or root system) that reaches the target location to execute its biological effect. The biological activity, off‐target toxicity, and side effects of agrochemicals are all influenced by their uptake, translocation, and redistribution in plants (Wu et al.,  2018). However, information about mode of pesticide behaviour in plants is relatively limited.

Many pests pierce plants by using specialized stylets and consume high amounts of fluids from the plant tissues, such as phloem and xylem, where all pesticides cannot reach easily. Suck‐feeding pests preferentially infest the underside of leaves or basal stem parts to avoid contact with foliar insecticidal sprays (Buchholz and Trapp, 2016). One of the most efficient chemical strategies to control insects that pierce and suck is the application of pesticides with vascular bundle mobility, which facilitates the ingestion of active ingredients by phytophagous pests (Wu et al.,  2018). Mathematical modelling of pesticide uptake and translocation behaviour in plants has emphasized the influence of hydrophobicity, water solubility, dissociation constant, pH of the external aqueous phase, and plant lipid content on pesticide use efficiency (Madikizela et al.,  2018; Miller et al.,  2016; Miller et al.,  2020). Approaches including the optimization of the physicochemical properties of active ingredients, adding of the adjuvant and nano‐crystallization of compounds have been used to improve membrane penetration and pesticide delivery in planta (Castro et al.,  2013; Chollet et al.,  2004; Yan et al.,  2020).

Recently, the role of plant‐membrane transporters in the exchange of signals and substances among cells, organs, and their environment has been extensively elucidated. Carrier mediation of agrochemical systemic distribution has shown to be a promising means for pesticide delivery in plants (Denis and Delrot, 1993; Chen et al.,  2018a, 2018b; Wu et al.,  2018). For instance, glucose or amino acid moieties have been introduced into insecticide molecules to produce phloem‐targeting drugs (Jiang et al.,  2009; Qin et al.,  2014; Yao et al.,  2017; Yuan et al.,  2013), partially because monosaccharide and amino acid transporters can regulate the pesticide conjugate uptake and translocation (Chen et al.,  2018a, 2018b; Mao et al.,  2017; Xie et al.,  2016). Furthermore, it has been reported that the broader substrate specificity of amino acid transporters (AATs) in plants facilitates the design and modulation of agrichemical transporter expression in plants via genetic manipulation towards the goal of precision pest management (Chen et al.,  2018a, 2018b; Ren et al.,  2019; Xie et al.,  2016).

The rice genome harbours at least 85 AAT genes, which mainly fall into two superfamilies: amino acid‐polyamine‐choline (APC) transporters and amino acid/auxin permeases (AAAP) (Taylor et al.,  2015). The AAAP super family comprise 58 genes, out of which eight distinct subfamilies are amino acid permeases (AAPs), lysine, histidine transporters (LHTs), proline transporter (ProTs), GABA transporters (GATs), auxin transporters‐like (ATL), aromatic and neutral amino acid transporters (ANTs), and amino acid transporters‐like (ATL) (Zhao et al.,  2012). To date, function of OsGATs has not been characterized, while some other members of the AAAP subfamily in rice have been well characterized. Most of the AAPs mediated amino acid transport with different affinities, and some of them are responsible for rice growth and development (Taylor et al.,  2015). Overexpression of OsAAP1, OsAAP4, OsAAP5, and OsAAP6 could change amino acid homeostasis in rice and enhance bud outgrowth, grain yield, and also grain protein content (Fang et al.,  2021; Ji et al.,  2020; Peng et al.,  2014; Wang et al.,  2019). However, blocking OsAAP3 expression results in decreased Arg, Lys, Asp, and Thr levels, and unexpectedly, promotes rice tiller and grain yield (Lu et al.,  2018). OsLHT1 is the only functionally characterized protein among the rice LHTs subgroup (Guo et al.,  2020; Guo et al.,  2020; Wang et al.,  2019). It functions in both root uptake and root‐to‐shoot allocation of a broad spectrum of amino acids in rice, similar to AtLHT1 in Arabidopsis thaliana (Chen and Bush, 1997). The ProT subgroup has only three members. OsProT1‐3 share nearly 40% of their amino acids, with Pro and GABA transportation ability and adaption to cadmium stress in rice plants (Lin et al.,  2019). All four members in OsANT subfamily did not show any transportation of amino acids in heterologous complementation experiments in yeast amino acid transporter mutant, but their expression in rice was changed in response to heat, salt, and cadmium stress, as well as nitrogen treatment (Xie et al.,  2021). OsAUX contains five members, and two of them have been functionally characterized. OsAUX1 and OsAUX3 exhibit auxin carrier activity; OsAUX1 also helps in response to cadmium stress and low external phosphate, while OsAUX3 responds to aluminium stress in rice (Giri et al.,  2018; Wang et al.,  2019; Yu et al.,  2015; Zhao et al.,  2015).

The amino acid transporter‐like (ATL) subgroup is thought to be involved in the compartmentalization of amino acids in the vacuoles of microorganisms and plants (Okumoto and Pilot, 2011). A survey of rice genome revealed 17 putative members of the ATL subgroup, which encode homologues of amino acid transporters in the vacuole of yeast (Saccharomyces cerevisiae) (ScAVTs) and Arabidopsis thaliana (AtAVTs) (Ogasawara et al.,  2021; Zhao et al.,  2012). They are divided into two subclades, ATLa (OsATL1–7) and ATLb (OsATL8–17) (Zhao et al.,  2012). OsATL6, which belongs to the ATLa subclade, was reported to be involved in the temporary storage of excessive glutamine in root vacuoles before being translocated to the shoot (Ogasawara et al.,  2021). OsATL14 (Bh4), which was isolated using a mapping–cloning approach, belongs to the ATLb subgroup and controls the black hull of wild rice (Oryza rufipogon); OsATL14 (Bh4) disruption results in the straw‐white colour of hull in cultivated rice, indicating phenotype selection during rice domestication (Zhu et al.,  2011). Overall, the physiological relevance of ATL subgroup in plants is mostly unknown.

Thiamethoxam (THX) is a neonicotinoid used extensively to control sucking insect pests, including the brown planthopper (BPH) (Nilaparvata lugens), which is an economically important rice pest in Asia (Nauen et al.,  2003). THX possesses several properties, including low molecular mass, high polarization and water solubility (4.1 g/L at 25 °C), and low partition coefficient [(log Pow) −0.13 at pH 6.8]. Consequently, it is rapidly and efficiently absorbed by the plant roots and leaves, with most of it being accumulated in the leaf tip via xylem (Maienfisch et al.,  2001). However, planthoppers mainly feed on the rice stem, resulting in low ingestion of THX (Buchholz and Trapp, 2016; Wu et al.,  2018). Therefore, characterization and understanding of transporters involved in THX uptake and translocation in rice would provide opportunity to relocate and accumulate THX at the planthopper feeding sites by genetic manipulation, thus improving the bioavailability of the insecticide.

In this study, we have characterized OsATL15 and studied its involvement in THX uptake and translocation in rice. Our results indicate that OsATL15 expression is associated with THX accumulation in rice tissues (roots, stems, and leaves). Expression of OsATL15 was controlled by a phloem‐specific expression promoter (gadcsPpro; Wang et al.,  2020a, 2020b) and could increase the accumulation of THX in rice stem, leading to a greater control over BPH damage. OsATL15 represents a potentially valuable gene in precise pest control that can be applied using biotechnology approaches.

Results

THX uptake and transport in rice plants

To determine THX uptake, rice seedlings were grown hydroponically for 28 day and then transferred to 0.5 mm CaCl2 solution containing THX. The results of the liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses showed that the THX content in the roots and stems of rice exposed to 100 μm THX for 24 h was approximately 500 nmol/g fresh weight (FW), whereas that of the leaf was approximately 1500 nmol/g FW. These results indicate a high THX translocation potential and the leaves being the primary THX accumulation tissue (Figure 1a).

Figure 1

Effects of different factors on thiamethoxam (THX) uptake in 21‐day‐old ZH11 rice seedlings. (a) Effects of inhibitors and darkness on THX uptake. CCCP—Carbonyl cyanide m‐chlorophenylhydrazone; DNP—2,4‐dinitrophenol; CK, only THX added. (b‐e) Comparison of THX content in rice plants under 28 or 4 °C in concentration gradient experiments. Three‐week‐old rice seedlings were cultured in 0.5 mM CaCl2 containing 50‐, 100‐, 200‐, 400‐, and 500 μm THX at 28 or 4 °C. THX content in the whole plants (b) root (c), stem (d), and leaf (e) was measured at different concentration points. Values are the means ± SD (n = 3). [Colour figure can be viewed at wileyonlinelibrary.com]

Carbonyl cyanide m‐chlorophenylhydrazone (CCCP) and 2,4‐dinitrophenol (DNP) have been widely used to study the transmembrane transport ability of plant transporters. They act as ATP synthesis inhibitors by disrupting the proton gradient across the membrane (Denis and Delrot, 1993; Honda et al.,  2012). Treatment of seedlings with 50 μm CCCP or 50 μm DNP significantly decreased THX concentrations in the roots, stems, and leaves, indicating that THX uptake was influenced by CCCP and DNP. Similarly, compared with control, there was a significant decrease in the THX content of seedlings under dark condition for 12 h, indicating that THX uptake could be increased by light (Figure 1a). These results suggest that the absorption and accumulation of THX in rice may occur via active transport, mediated by the availability of energy source and facilitated by transporters.

To further explore the net uptake of THX mediated by the active transport process, we performed THX uptake experiments by maintaining a THX concentration gradient at 4 °C and 28 °C. The results showed that the THX content in the whole plant at 28 °C was higher than that at 4 °C (Figure 1b). Notably, more THX accumulation occurred in stem at 4 °C than at 28 °C, whereas the opposite trend was observed in leaves and roots (Figure 1c‐e). Overall, we inferred that THX transport occurs via active transport; however, whether it is the main route is unclear.

Screening of a rice‐transporter‐enriched yeast expression library revealed a THX transporter

Full‐length cDNAs (1358 bp) encoding transporter genes in rice collected from Rice Genome Resource Center, Tsukuba, Japan, have been successively subcloned into yeast expression vectors pYES2 and transferred into yeast strain W303‐1A (Yamaki et al.,  2017). The resulting library was used to identify transporters involved in THX uptake and accumulation. A preliminary assay showed that 4 and 10 mm THX slightly and severely inhibited the growth of yeast cells carrying pYES2 empty vector, respectively (Figure 2a), suggesting that THX is toxic to yeast cells. We postulated that the growth of yeast cells carrying rice transporters would be inhibited more severely because of the increase in THX uptake compared with the control strain without rice transporters. Therefore, colonies sensitive to THX were screened using SD‐Gal agar medium containing either 4 or 10 mm THX, resulting in the isolation of a THX‐sensitive clone expressing OsATL15 [AK242293 in Kikuchi et al.,  2003, LOC_Os01g41420 from http://rice.plantbiology.msu.edu/analyses.shtml] (Figure 2a and S1). This result was further supported by the significantly higher THX content in cells expressing OsATL15 compared with the cells containing the empty vector (Figure 2b).

Figure 2

Identification of thiamethoxam (THX) transporters by screening a membrane‐gene‐enriched expression yeast library. (a) Growth of yeast strains carrying OsATL15 gene on SD‐Glc solid media containing 4 mm or 10 mm THX. The medium containing 1% DMSO was set up as a control. (b) THX uptake assay in yeast. Transformations carrying the empty pYES2 vector were set up as a control. Data are mean ± SD of biological replicates (n ≥ 3). Statistical comparison was performed using Tukey's test (**P < 0.01). [Colour figure can be viewed at wileyonlinelibrary.com]

Rapid amplification of cDNA ends (RACE) experiments and Protter predictions (http://wlab.ethz.ch/protter/start/) showed that OsATL15 encodes a protein of 426 amino acids and 10 transmembrane domains (Figure S1a). Phylogenetic analysis was performed to examine the evolutionary relationships among the ATL proteins in A. thaliana, Oryza sativa, and Zea mays. The results showed that the ATLb proteins in rice were primarily divided into two clades, with OsATL12 to OsATL17 belonging to one clade. OsATL15 exhibited high amino acid sequence similarity (84% identity) to Z. mays L. GRMZM2G066428.01, but its homologue was not identified in A. thaliana (Figure S1b).

facilitates uptake and accumulation of THX in rice

To further explore the role of OsATL15 in THX uptake and translocation in rice, OsATL15 knockout mutants (osatl15‐1 and osatl15‐2) and overexpressing lines (OX‐2 and OX‐17) were generated using ZH11 as the genetic background (Figure S2). Lines containing various OsATL15 genotypes were exposed to 100 μm THX for 1, 2, 3, 6, 12, 18, and 24 h. Overall, THX uptake increased gradually with an increase in treatment duration. Compared with wild type, there was a 49.74%, 74.69%, and 65.46% decrease in the THX content of the roots, stems, and leaves of OsATL15 knockout mutants, respectively. In contrast, there was a 197.05%, 151.53%, and 251.90% increase in the THX content in the roots, stems, and leaves of the lines overexpressing OsATL15 compared with that in the wild‐type (WT) plants (Figure 3 and Table S1).

Figure 3

Thiamethoxam (THX) in planta content varies across OsATL15 genotypes. Three‐week‐old seedlings were cultured in 0.5 mm CaCl2 containing 100 μm THX. THX content in the root (a), stem (b), leaf (c), and whole rice plants (d) of OsATL15 transgenic lines and the wild type (WT) were measured at 1‐, 2‐, 3‐, 6‐, 12‐, 18‐, and 24‐h time points. Data are mean ± SD of biological replicates (n ≥ 3). FW—fresh weight. [Colour figure can be viewed at wileyonlinelibrary.com]

To further investigate the role of OsATL15 in THX uptake, the THX content in the roots, stems, and leaves of rice seedlings was observed over a concentration gradient following 12 h of exposure at 28 and 4 °C (Figures 4 and S3). The results showed that there was no significant difference in the THX uptake of lines containing various OsATL15 genotypes at 4 °C (Figure S3). However, the THX content in OsATL15‐overexpressing lines was mostly higher than that in lines carrying mutant osalt15 at 28 °C (Figure 4a‐d). Moreover, there was a 156.46%, 153.91%, and 182.17% increase in the THX content in the roots, stems, and leaves of OsATL15‐overexpressing rice seedlings, respectively, compared with that of ZH11 rice. In contrast, there was a 60.40%, 52.67%, and 40.72% decrease in the THX content of the roots, stems, and leaves of osatl15 mutants, respectively, compared with that of ZH11 rice (Figure 4a‐d and Table S2).

Figure 4

Thiamethoxam (THX) uptake kinetics in OsATL15 rice genotype. Contents of THX in roots (a), stem (b), leaf (c), and whole rice plants (d) in OsATL15 genotype lines and wild‐type (WT) at 28 °C. (e) Net uptake of THX was calculated by THX contents in rice at 28 °C minus those at 4 °C. Curves represent the fitted Michaelis–Menten equations, with K and V max shown next to the curve. Data are means ± SD of three biological replicates. FW—fresh weight. [Colour figure can be viewed at wileyonlinelibrary.com]

Net THX uptake was further calculated by subtracting the THX content at 4°C from that at 28°C. A graph of net THX uptake against THX concentrations showed that THX uptake in various OsATL15 genotypes saturated to varying degrees under higher THX culture concentrations. In the OsATL15 overexpression lines, the highest uptake rates were 457.1 and 420.4 nmol/g/h, whereas those of the osatl15 mutant lines were only 130.7 and 92.19 nmol/g/h. These results further confirmed the role of OsATL15 in THX uptake in rice (Figure 4e).

Cellular and tissue localization of OsATL15

The full‐length coding sequence of OsATL15 was fused to the N terminus of green fluorescent protein (GFP) under the control of cauliflower mosaic virus (CaMV) 35S promoter and transiently expressed in protoplasts isolated from etiolated rice seedlings. The OsATL15‐GFP fluorescence signal was observed in the plasma membrane, showing co‐localization with mCherry‐1008, a known plasma membrane marker. In contrast, the GFP signal of the control (GFP alone) was detected in both the cytosol and nucleus (Figure 5a). Overall, subcellular localization analysis confirmed that OsATL15 is localized in the plasma membrane of rice cells.

Figure 5

Subcellar localization and tissue distribution of OsATL15. (a) Subcellular localization of OsATL15 in rice protoplasts. OsATL15 fused with green fluorescent protein (GFP) at N‐terminal (row 1) or GFP alone (row 2) was transiently co‐expressed with plasma membrane marker 1008‐mCherry in rice sheath protoplasts by PEG‐mediated transformation and visualized by confocal microscopy. GFP signals, mCherry signals, bright field image, and merge image are shown from column left to right. At least 20 individual cells were analysed for each localization experiment. Scale bar = 10 μm. (b) Histochemical staining for β‐glucuronidase (GUS) activity in transgenic plants expressing a OsATL15 pro:GUS construct. GUS activity in main roots, stems, leaves, and seeds is shown in column 1. Bar = 5 mm. Column 2 shows cross section derived from column 1. Bar = 50 μm. (c) Distribution of OsATL15 immunoreactivity in rice tissue. Immunostaining using an OsATL15‐specific antibody was performed on tissue of 30‐day‐old seedlings (row 1 and 2), and immunostaining using a β‐glucuronidase (GUS) antibody was performed on the heading‐stage plants (row 3 and 4). Bars = 100 μm. Row 2 shows enlarged images of the yellow box area corresponding to row 1; row 4 shows enlarged images from yellow dotted box corresponding to row 3. Bars = 20 μm. The red colour shows signals from the antibody; cyan blue is derived from autofluorescence of the cell wall and nuclei stained by DAPI. en—endodermis; ep—epidermis; p—phloem; x—xylem vessel. [Colour figure can be viewed at wileyonlinelibrary.com]

We further determined OsATL15 distribution patterns in tissues and cells based on histochemical analyses. Stable transgenic rice lines expressing the β‐glucuronidase (GUS) reporter gene driven by the OsATL15 gene promoter (1968‐bp from the start codon) were generated. Histochemical GUS staining of 30‐day‐old seedlings showed that OsATL15 was highly expressed in the roots, stems, and leaf blades, but not in the root caps (Figure 5b). GUS activity was high in root transverse sections, stem longitudinal sections, and leaf blade vascular bundles (Figure 5b, row 1–3). In contrast, GUS activity was not detected in hulls or in brown rice at filling stages (Figure 5b, row 1–3).

Root, stem, and leaf samples were cross‐sectioned and immunostained using both an anti‐OsATL15 polyclonal antibody (for 30‐day‐old seedlings) and a GUS‐specific antibody (for plants in the heading stage). The images revealed that the GUS signals from OsATL15 were localized in both the exodermis and endodermis and in the xylem Parenchyma cells with expanded vascular bundles in rice roots (Figure 5c, column 1). These findings revealed that OsATL15 was found in xylem parenchyma cells, comprising the transfer cells of the enlarged vascular system of the stem, epidermis, and parenchyma sheath in leaves (Figure 5c, column 3).

Tissue‐specific expression of enabled higher accumulation of THX in vascular bundles

Studies have established that BPH feeds on vascular bundles, especially the phloem. To enhance the THX accumulation in the tissues where the BPH feeds, rice lines expressing OsATL15 under the control of a vascular‐specific promoter gdcsPpro (Wang et al.,  2020a, 2020b) were generated. GUS staining in roots, stems, and leaves showed that gdcsPpro promoter allowed OsATL15 expression in xylem, phloem, pericycle, and endodermis in roots, as well as in vascular bundles of stems and leaves (Figure 6a).

Figure 6

Vascular‐specific expression of OsATL15 increased THX content in xylem sap in rice. (a) Histochemical staining for β‐glucuronidase (GUS) activity in transgenic plants expressing gdcsPpro::OsATL15‐GUS construct. GUS activity in the main roots, stems, leaves, and their cross sections is shown. (b) A presentation of vascular bundle tissues cut by Laser capture micro‐dissection (LMD). (c) A total of 12 mm2 samples cut by LMD for each line were combined for THX content quantification by LC–MS analysis. (d) THX content in xylem sap collected from OsATL15 transgenic lines and the WT. (e) Transcription level of OsATL15 in vascular bundles tissue derived from OsATL15 transgenic lines and the WT. Data are mean ± SD of biological replicates (n ≥ 3); Different small letters indicate significant difference at P < 0.05 by Tukey's test. [Colour figure can be viewed at wileyonlinelibrary.com]

The THX content of the vascular bundles of the stems and leaves in various OsATL15 genotypes lines was determined using laser micro‐dissection (LMD) technology following LC–MS/MS detection (Figure 6b). The results revealed that OsATL15 expression could influence THX contents in rice vascular bundles. THX levels in the BPH feeding tissues were the highest in gdcsPro::OsATL15‐1 plants, followed by OX‐17 line (overexpressing OsATL15), and lower in osatl15‐2 mutant lines compared with those of the WT plants (Figure 6c). These observations were further confirmed by determining THX content in xylem sap collected from various OsATL15 genotype lines (Figure 6d). Furthermore, quantitative real‐time (qRT)‐PCR also confirmed that OsATL15 expression levels in vascular tissue cut by LMD were positively correlated with THX contents in stems across the genotypes (Figure 6e). Overall, THX content increased with the increase in OsATL15 under low THX concentration.

overexpression and vascular‐specific expression resulted in higher effective BPH control by THX

To test whether changes in THX content in different OsATL15 genotypes could influence pest control effectiveness, BPH mortality in three OsATL15 genotypes at three growth stages (12, 45, and 70 days after sowing, the periods in which BPH caused severe damage) were calculated under 30 and 100‐μm THX root exposure conditions, to simulate different field soil application scenarios (Figure 7a). In all the treatments, the corrected mortality rates of BPH on OX‐17 plants were significantly higher than those of BPH on WT plants, whereas the mortality rates of BPH on osatl15 mutant lines were lower than those in the two former conditions. Additionally, there was a slight decrease in BPH mortality rate with the increase in plant age across the genotypes (Figure 7b‐c, Table S3).

Figure 7

Changes in thiamethoxam (THX) content in OsATL15 genotype lines and their toxicity against brown planthopper. (a) Schematic diagram of a hydroponics pot with rice seedlings for testing the toxicity of THX against brown planthopper. Corrected mortality (%) of 30 μm (b) and 100 μm THX (c) to BPH on 12‐, 45‐, and 70‐day‐old OsATL15 genotype strains, WT, knockout (osatl15), overexpression (OX‐17), and vascular bundles specific expression lines (gdcsPpro::OsATL15). Representative images of 15‐day‐ (d), 45‐day‐ (e), and 70‐day (f)‐old seedlings of OsATL15 genotype lines after root uptake of 30 μmol of THX for 24 h and infestation with BPH. Photographs were taken after 4 days, 8 days, and 15 days of insect infestation of 12‐, 45‐, and 70‐day‐old seedlings, respectively. Data are mean ± SD of biological replicates (n ≥ 3); different small letters indicate significant difference at P < 0.05 by Tukey's test. Bars = 10 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Under the 30‐μm THX condition, the mortality rate of BPH on WT plant was 19.15%–23.77%, whereas that on plants overexpressing OsATL15 was 43.31%–72.59% across all examined growth stages (Figure 7b, Table S3). Under 100 μM THX, the mortality rate of BPH on plants overexpressing OsATL15 ranged from 65.03% to 92.78%, whereas that on osatl15 mutant plants ranged from 44.19% to 51.67% (Figure 7c, Table S3), indicating that the increase in THX concentration from 30 to 100 μm (about threefold) enhanced BPH control. However, although BPH mortality rate increased with an increase in THX concentration, the degrees of improvement in efficiency were not commiserative with degrees of increase in concentration, suggesting a THX uptake saturation point (<100 μm) in rice. Therefore, we conclude that the overexpression of OsATL15 could adequately improve the efficiency of THX to control BPH under field THX application concentrations.

The effectiveness of THX in controlling BPH on OsATL15 genotype lines was further demonstrated as symptoms of infestation were reduced. Seedlings of the three growth stages (12, 45, and 70 days) were planted in a pot containing soil. After 24 h of 30 μmol THX application, plants were inoculated with BPH. Photographs were taken until the mock controls (without THX application) died. The results showed that 30 μmol THX provides protection to OsATL15‐overexpressing, gdcsPro::OsATL15‐1 lines, as well as wild type, but less protection to osatl15 knock out lines (Figure 7d‐f). These results suggest that the expression of OsATL15 enhances THX accumulation, thus providing better protection against BPH.

Discussion

The improvement of the efficiency of chemical pesticide use has been the subject of most modern agricultural research. Several attempts have been made to improve pesticide phloem mobility by optimizing the structure and physicochemical properties of chemicals (Chen et al.,  2018a, 2018b; Kleier and Hsu, 1996). However, the application of such method is limited by the decreased entomotoxicity and complex synthesis procedures (Yao et al.,  2017). Here, we provided evidence that OsATL15 plays an essential role in systematic distribution of THX in rice plants, indicating that OsATL15 could be a valuable gene in breeding programme to improve target application and efficiency of the pesticide.

The analyses of uptake kinetics, tissue distribution, and localization results indicate that OsATL15 is involved in THX root uptake and transfer in rice. The amount of THX that entered the roots and translocated to the leaves in rice plants varied across OsATL15 genotypes; additionally, OsATL15 was found to be localized in the exodermis, endodermis, and the xylem parenchyma cells of enlarged vascular bundles of the root meristem, indicating that OsATL15 participates in THX transportation in rice. However, knockout of OsATL15 and decreased temperature did not completely inhibit THX mobility in rice. This result suggests that despite the critical role of OsATL15 in THX uptake in rice roots and accumulation in leaves, other factors influence the systemic distribution of THX in rice. Alternatively, OsATL15 may also be involved in THX redistribution in rice, as plants have developed complex homoeostatic networks for the maintenance of the concentrations of foreign organic compounds such as THX in shoots. OsATL15 was highly expressed in the vascular bundles of stems and leaves, implying that it played a role in THX distribution. Rice roots are characterized by two Casparian strips in both the exodermis and endodermis, between which almost all the cortex is destroyed. Therefore, the rice root xenobiotic uptake system is mediated by influx and efflux transporters that work synergistically (Sasaki et al.,  2012). Substance distribution in rice is skewed towards the expanded leaves, with subsequent redistribution to developing tissues via phloem‐kickback (Wang et al.,  2020a, 2020b). Therefore, identifying other mechanisms of THX transport and stem allocation and associated genes is a prerequisite for breeding rice cultivars with optimized insecticide use efficiency.

BPH can cause serious damage to rice by sucking rice phloem sap, ovipositing in rice tissues, and transmitting viral rice diseases, which are especially harmful at the vegetative stage (Maienfisch et al.,  2001). The present study showed that OsATL15 expression, THX concentrations, and BPH mortality have consistent trends in both space and time. Compared with WT or osatl15 mutant lines, there was a significant increase in THX concentration in the stem vascular bundles of OsATL15‐overexpressing plants, which increased BPH mortality by approximately 20%–30% in a modified BPH bioassay of rice grown in a greenhouse.

Expression of OsATL15 under the control of vascular bundle‐specific promoter, gdcsP, led to THX accumulation in the vascular bundles in rice and increased BPH mortality successfully. The expression of the portions (e.g. Insecticidal or virus) is extremely efficient in the development of genetically superior lines with increased resistance capability to hemipteran insects, which suck phloem sap and some viruses, that exclusively replicate or interact with phloem cells (Wang et al.,  2020a, 2020b). Therefore, THX application could be reduced by manipulating the expression of OsATL15. Furthermore, GUS assay showed that OsATL15 was not expressed in the edible parts of rice, such as glume and seeds. Although overexpression or phloem‐specific expression of OsATL15 caused accumulation of THX and enhanced its BPH control, THX residue could not be detected in the seeds, indicating that OsATL15 could regulate THX accumulation at the tissue damage site by BPH, thus reducing the risk of pesticide residues in edible plant parts.

Although the OsATL family has the second most members in the rice AAAP superfamily, their functions are still largely unclear and there is no evidence that they transport amino acids. A Conserved Domain Database search across multiple species OsATL15 is a member of the SLC5‐6‐like_sbd superfamily, which also includes the solute‐binding domain of the SLC5 proteins (also known as the glucose/sodium co‐transporters or solute sodium transporters), SLC6 proteins (also known as the sodium and chloride–dependent neurotransmitters or Na+/Cl–‐dependent transporters) and nucleobase–cations–symport–1 (NCS1) transporter (Lu et al.,  2020; Schlessinger et al.,  2010). The SLC6 family is noteworthy within the SLC families, consisting of 20 genes encoding transporter proteins with conserved functional domain. These proteins are capable of transportation of amino acids and amino acid derivatives into cells in and against chemical gradients means, by using the driving force involved in the co‐transporting of extracellular Na+ as a for substrate (Bröer, 2006; Chen et al.,  2004; Horiuchi et al.,  2005). SLC6 transporters are separated into four inferior levels on account of sequence similarity and substrate specificity: amino acid, monoamine, GABA, and amino acid/orphan (He et al.,  2009; Kristensen et al.,  2011). We noted sequences in the rice ATL family that encode proteins similar to unc‐47 in Caenorhabditis elegans and Avt1p in Saccharomyces cerevisiae; these belong to the SLC6 transporter family (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The unc‐47 protein is expressed by GABA neurons and confers vesicular GABA transport, and the yeast Avt1p transporter function has been observed to control amino acid flux in vacuole‐like organelles (Russnak et al.,  2001). OsATL15 can transport THX, a well‐known insecticide which disrupts the function of nicotinic acetylcholine receptors (nAchRs) in the central nervous system of insects (Nauen et al.,  2003). Thus, similarly, it can be speculated that OsATL15 acts as a natural neuronal substrate for neurotransmitter transport in plants. Although it has been reported that the SLC6 family does not exist in plants, a growing number of studies have found that plants have neurotransmitters, capable of transmitting physical and chemical signals (Chao et al.,  2021). For example, Nicotiana attenuata accumulate nicotine when it is damaged by the hornworm Manduca Sexta, which suppress acetylcholine receptors and, therefore, is toxic to the insect that depend on neuromuscular junctions (Baldwin, 2001). Plants seem to trigger neuron‐like cell–cell transmission by acetylcholine as also they produce neuronal acetylcholinesterase (Sagane et al.,  2005； Momonoki et al.,  1998). Besides, although our results clearly show that OsATL15 protects plants from BPH damage via THX uptake in great amounts, whether it is involved in BPH resistance is still unclear. Therefore, relationships among OsATL15, neurotransmitters, and BPH resistance need to be further explored.

In summary, ectopic expression of OsATL15 increases the accumulation of THX in rice stems, improving its effectiveness against BPH. OsATL15 is a potentially valuable gene for breeding rice cultivars with efficient pesticide utilization and potential critical environmental benefits.

Experimental procedures

Chemicals and materials

THX active compound and its 30% formulation were purchased from Haoyang Chemical Co., LTD. (Hebei, China) and Jianpai Agrochemical Co., LTD. (Jiangsu, China), respectively. The other reagents used in the study were purchased from Sigma‐Aldrich Chemical Corporation, Ltd. (St. Louis, MO, USA). Primers were synthesized by Tianyihuiyuan Biotechnology (Beijing, China) and are listed in Table S4.

Plant growth conditions

Seeds were soaked in 2% sodium hypochlorite for 15 min, washed twice using distilled water, and stored at 37 °C for 1 day and placed in petri dishes containing two layers of wet filter papers until the primary roots of immature embryos grew to 2 cm in length. Seedlings were transferred to a tank with a set of plastic mesh and modified Hoagland nutrient solution and grown in the chamber under a 12‐h night/day photoperiod, light intensity >350 μmol/m2/s, day/night temperatures of 30/22 °C, and relative humidity of 60% for time points for THX root uptake experiments and BPH bioassay. The nutrient solution was renewed every 3 days.

Yeast expression assay

A rice transporter‐gene‐enriched yeast expression library was used to identify the transporters involved in THX uptake following previously described procedures (Yamaki et al.,  2017). Briefly, appropriate concentrations of THX for use in yeast library screening were determined by culturing yeast carrying an empty vector (pYES2) on SD‐Gal containing 1–10 mm THX. Yeast cells were recovered for 2 days at 30 °C and were harvested and diluted to an OD600 value of 0.01 with distilled water, and 10 μL of the cultures were spotted on SD‐Gal (2% galactose) solid medium containing either 4 or 10 mm THX, using a Bell Blotter 96‐well replicator (Tech‐jam Co., Osaka, Japan). The plates were incubated at 30 °C for 2–3 days before being photographed.

To determine THX uptake into the yeast cells, the overnight cultures were centrifuged at 2000 g for 5 min and the supernatant was discarded. A total of 5 mL of fresh SD‐Gal was then added, and the cells were cultured for 3 h at 30 °C. Approximately, 5 μL of 50 mm 5‐fluorouracil was added and the cells were cultured for 1 h to stop cell growth, so that a cell density of 1 × 107 cells (100 μL)−1 was obtained, which was determined using a cell counter. Diluted cells were then shake‐cultured for 1 h at 400 g and then maintained at 1200 g for 2 min. Thereafter, 0.333 mm MES‐NaOH buffer (100 μL; pH 5.5) containing 2% glucose, 4 mm THX, and 50 μm 5‐fluorouracil was then added to each culture and shaken at 3000 rpm for 1 h at 30 °C. After harvesting by centrifugation at 2000 g and 30 °C and washing five times with ice‐cold deionized water, all cells were completely homogenized in 1 mL acetonitrile for 2 min and then ultrasonicated for 30 min. The mixture was then centrifuged at 2000 g for 10 min to acquire the supernatant for subsequent ultra‐performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) analysis.

Phylogenetic analysis and sequence alignment

The amino acid sequences of OsATL15 in the present study were extracted from the NCBI (https://www.ncbi.nlm.nih.gov/) databases. A phylogenetic tree was constructed in MEGA‐X (http://www.megasoftware.net/) using maximum‐likelihood phylogenetic analysis, with 1000 bootstrap replicates. Potential transmembrane domains of OsATL15 protein sequences were identified using Protter (http://wlab.ethz.ch/protter/start/).

Subcellular localization of OsATL15

To investigate the subcellular localization of OsATL15, the optical reading frames of OsATL15 without the stop codon were cloned into the pYL322‐EGFP‐N1 vector to obtain a construct containing N‐terminal GFP fusions, named OsATL15‐GFP. The plasmid OsATL15‐GFP or GFP alone (control), along with 1008‐mCherry, was then introduced into the rice protoplasts, which was isolated using the polyethylene‐glycol method (He et al.,  2016). Fluorescence signals were detected using a confocal laser scanning microscope (Leica SP8, Bensheim, Germany). Excitation/emission wavelengths were 488/506 to 538 nm for GFP and 561/575 to 630 nm for red fluorescent protein.

Histochemical GUS staining

For GUS staining, all tissues of 3‐week‐old hydroponically grown rice seedlings were incubated separately using a GUS solution kit (Coolaber, Beijing, China), according to the manufacturer's instructions, and reactions were performed overnight at 37 °C. Various plant tissues were harvested and fixed in buffer containing 4% (w/v) paraformaldehyde, 0.1 mmol/L sodium phosphate (pH 7.0), 0.25% (v/v) glutaraldehyde, and 0.1% (v/v) Tween‐20. The samples were dehydrated in a series of increasing concentrations of ethanol. Dehydrated products were then infiltrated and embedded in Epon‐812 resin. The polymerization reaction was performed overnight at 60 °C. Semi‐thin (5 μm) sections were prepared using an ultramicrotome (Leica UCT) and examined and photographed under a microscope (ZEISS Axio Observer D1, Gottingen, Germany).

Immunohistological staining

The synthetic peptide C‐DGSIASYPDIGQYAFG (positions 94 to 109 in OsATL15 deduced amino acid sequence) was used to immunize rabbits to obtain antibodies. The antiserum was purified through a peptide affinity column before use. Twenty‐one‐day‐old rice seedlings were used for anti‐ATL15 immunostaining. Roots, stems, and leaf blades were cut into small pieces (1–5 mm) with a razor blade and immediately put into freshly prepared fixative solution containing 4% (w/v) paraformaldehyde, 0.1 mmol/L sodium phosphate (pH 7.0), and 0.1% (v/v) Tween‐20. A vacuum was applied to the samples for 30 min and further fixed overnight at 4 °C. After fixation, samples were dehydrated in an ethanol gradient and then infiltrated and embedded in LR White resin (Ted Pella Inc., Redding, CA, USA). The polymerization reaction was performed by polymerizing the closed capsules under UV light for 48 h at 4 °C. Semi‐thin 1‐μm sections were prepared using an ultramicrotome (Leica UCT) (Avci et al., 2012), placed on microscope slides, re‐incubated in PBS containing 0.3% (v/v) Triton X‐100 at 30 °C for 2 h, washed three times with PBS, and blocked with 5% (w/v) bovine serum albumin in PBS. Slides were incubated in a 4 °C chamber overnight with the purified rabbit anti‐OsATL15 polyclonal antibodies (1:100 dilution in PBS). Thereafter, the slides were washed thrice using PBS, blocked with 5% (w/v) bovine serum albumin, incubated with secondary antibodies (Alexa Fluor 555 goat anti‐rabbit IgG; BBI) for 2 h with a dilution of 1:200 at 25 °C, washed five times in PBS in the dark, and mounted with anti‐fluorescence quenching sealing liquid (including 4′,6‐diamidino‐2‐phenylindole [DAPI]) (Beyotime, P0131, China). To detect the GUS protein under the control of OsATL15 promoter in transgenic rice tissues, a rabbit anti‐GUS polyclonal antibody (Agrisera, AS16, 3689) was used as the primary antibody for immunostaining. Alexa Fluor 555 goat anti‐rabbit IgG was used as the secondary antibody, with a dilution of 1:200 (Yamaji and Ma, 2014). Sections were examined under a laser scanning confocal microscope (TCS SP8x, Leica Microsystems).

Hydroponic exposure experiment

Hydroponic exposure experiments were performed in 50‐mL tubes containing 10 hydroponic seedlings for two dependent lines of each OsATL15 genotype (3‐week‐old). The tubes were wrapped with foil paper, and the open areas between the cap and the rice seedlings were sealed with sponge wrapped with foil paper. The rice roots were exposed to 0.5 mm CaCl2 (40 mL) containing THX over a concentration gradient (50‐, 100‐, 200‐, 300‐, 400‐, and 500‐μm THX) for 12 h at 28 and 4 °C. To determine the effect of the duration of THX exposure on its uptake and accumulation, the roots were exposed to 100‐μm THX for 1, 2, 3, 6, 12, 18, and 24 h. In inhibition experiments, metabolic inhibitors (50‐μm CCCP or 50‐μm DNP) were added to each tube for 6 h before sample collection, and tubes filled with solution without inhibitors were used as controls. In dark experiments, rice seedlings were treated in the dark or exposed to light for 12 h each. All experiments were carried out in triplicate. After THX exposure, the plants were harvested and thoroughly macerated in liquid nitrogen and 10 mL acetonitrile was added to the stems and leaves, while 5 mL acetonitrile was added to the roots. The mixtures were shaken vigorously for 2 min using a shaker and ultrasonicated for 30 min. Subsequently, 4 and 1 g of anhydrous MgSO4 and NaCl, respectively, were added, and the mixtures were vortexed for 1 min. The mixtures were then centrifuged at 1000 g for 5 min to acquire the supernatant. After this, 1.5 mL of the upper layer was transferred to a 2‐mL centrifuge tube and cleaned with 50 mg of PSA (for THX), 250 mg of MgSO4, and 10 mg of graphitized carbon black (GCB, but no GCB for the roots). The resulting supernatant was filtered through 0.22‐μm nylon syringe filters for UPLC–MS/MS analysis.

To measure THX concentration in xylem sap in rice, the 30‐day‐old plants were exposed to 100 μm THX in the nutrient solution as the method mentioned above for 1 day. The shoots were excised at 4 cm above the roots with a razor. Xylem sap was collected from the cut surface for 2 h using a micropipette according to the method described by Yokosho et al. (2009).

UPLC–MS/MS analysis was carried out by using an Acquity TQD UPLC–MS/MS (Waters Corp., Milford, MA, USA). Chromatographic separation was carried out with an ethylene bridged hybrid C18 column (50 × 2.1 mm; 1.7 μm) at 25 °C and 1‐μL injection volume as previously described (Maienfisch et al.,  2001, Table S3).

Vector construction and generation of transgenic plants

To confirm the length of transcript of OsATL15, 3′‐rapid amplification of cDNA ends (3′‐RACE) was performed using a Smart RACE cDNA amplification kit (Takara, Dalian, China). The ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) was used for vector construction. The 1278‐bp OsATL15 open reading frame was produced from cDNA derived from ZH11, and inserted downstream of the 35S promoter in pCAMBIA1300‐35S, generating a 35S::OsATL15 expression cassette, and was used to transform wild‐type ZH11 via Agrobacterium tumefaciens‐mediated transformation. T1 seeds originating from 17 individual lines were collected, and ten of them were screened separately for hygromycin resistance and gene expression level analysis, leading to two dependent lines (Ox‐2 and Ox‐17) for phenotypic analysis (Figure S2a). The method of establishing CRISPR‐Cas9 knockout lines of osatl15 has been described previously (Ma et al.,  2015). The T0 and T1 homozygous of two dependent mutants (osatl15‐1 and osatl5‐2) were detected by sequencing PCR products produced by primer pairs flanking two OsATL15‐specific target sites (Figure S2b). For the GUS constructs, a 1968‐bp sequence preceding the translation initiation site of the OsATL15 was amplified using genomic DNA from cultivar Zhonghua11 as template and inserted into the pCAMBIA1300‐GUS vector to form a cassette expressing OsATL15pro::GUS. To generate OsATL15 vascular‐specific expression lines, a putative 5000‐bp gdcsP promoter (Wang et al.,  2020a, 2020b) and coding region of OsATL15 were assembled in pCAMBIA1300‐GUS and pCAMBIA1300 vectors using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China), yielding gdcsP::OsATL15‐GUS and gdcsP::OsATL15 recombinant constructs, respectively. The sequences of all constructs were confirmed by DNA sequencing before transformation into cultivar ZH11 to obtain stable transgenic rice lines.

Bioassay of THX against rice planthopper in greenhouse

Our laboratory strain of BPH was originally collected from natural populations in the South China Agricultural University (Guangzhou, China) research farm. The BPH colony was allowed to multiply for two generations in an insect‐rearing cage (45 cm × 45 cm × 50 cm) on ZH11 rice seedlings in the laboratory without exposure to insecticides at 26 ± 1 °C under a 16:8‐h light:dark photoperiod with 70%–80% humidity. The host plants were replaced weekly, and only second‐ to third‐instar nymphs were selected for bioassay. One hundred BPH individuals were placed in a 10‐mL test tube using a small hand‐held vacuum cleaner (ZK‐XWQ; Zhike, Zhengzhou, China).

A modified version of the rice seedling dip method (He et al.,  2011) was used to examine systemic toxicity of THX against BPH on one line of each OsATL15 genotype (osatl15‐1, OX‐17, and gdcsP ::OsATL15‐1). Hydroponic seedlings at three growth stages (12, 45, and 70 days old) were subjected to preculture in 30‐μM (or 100‐μm) THX solution for 24 h in a 700 mL plastic bubble tea cup that has been specially adapted for monitoring BPH mortality. The cover of the cup was placed upside down, and its centre hole was used for inserting a planting cup with a dry cotton surrounding at the bases of the rice stems (Figure 6a). After 24 h of THX uptake, 30 third‐instar nymphs were inoculated to the seedlings in each cup and placed in a zippered plastic bag for 48 h before recording mortality. The insects that fell on their back and were unable to recover normal posture were counted as dead. The bioassay was conducted at 26 ± 1 °C, under 70%–80% relative humidity and a 16:8‐h light:dark photoperiod. Plant couture in 0.5 mm CaCl2 solution supplemented with 0.1% dimethyl sulfoxide (DMSO, Sigma Aldrich, Milwaukee, WI, USA) was set up as the control group. Mortality data were corrected using the data from the 0.5 mm CaCl2 solution and control treatment (0 μm THX) based on the Abbott formula (Abbot, 1925). Each treatment contains three replicates, and the bioassay was repeated thrice.

To phenotype the THX against BPH feeding on each line, rice seedlings were grown in the plastic pots (diameter, 26 cm; height, 20 cm) with paddy soil for 12, 45, and 70 days. After 24 h of root application, 1000 mL of 30 μm THX was added to each pot, and the 12‐, 45‐, and 70‐day‐old seedlings were infested with second‐ or third‐instar BPH nymphs at a density of 30, 300, and 1500 nymphs per seedlings, respectively. Severe damage symptoms were observed, and photograph of 12‐, 45‐, and 70‐day‐old seedlings was taken after 3, 7, and 20 days of insect inoculation, respectively.

Laser capture micro‐dissection

Rice leaves and stems from the hydroponically cultured seedlings were cut into small pieces (approximately 1–1.5 cm long) and embedded in cryogel matrix (Leica Microsystems, Bensheim, Germany) for cryo‐sectioning. Root samples were cut into 25‐μm thick sections using a cryotome (Leica CM1950, Bensheim, Germany) and carefully placed on metallic non‐fluorescent polyethylene terephthalate slides. Vascular bundle tissues from stems and leaves were micro‐dissected using a laser capture micro‐dissection system (Leica LMD7000, Bensheim, Germany) set at 349 nm wavelength, power of 56 μJ, pulse frequency of 1695 Hz, and 20× magnification. The tissues fell into caps of 500‐μL micro‐centrifuge tubes by gravity; the combined areas were 12 mm2 for each sample. The separated tissue parts in each cap were transferred to the bottom of the tube by centrifuging for 10 min (13800 g, 17 °C). Acetonitrile (100 μL) was added into each collection tube and sonicated for 60 min and then centrifuged again for 10 min (10 000 g, 17 °C). The supernatant (90 μL) was transferred into a glass insert with a plastic bottom spring in a 1.5‐mL brown HPLC grade vial and stored at 4 °C before UPLC–MS/MS analysis (Chen et al.,  2018a, 2018b; Jaiswal et al.,  2018).

Statistical analysis

One‐way analysis of variance followed by Tukey's test was performed to compare significant differences (P < 0.05) using IBM SPSS Statistics 20 for Windows (IBM Crop., Armonk, NY, USA). Means and standard deviations were calculated using triplicate measurements.

Conflict of interest

The authors have no conflict of interest to declare.

Author contributions

The research was conceived and supervised by F.L. and H.H.X.; Y.Y.X., F.L., H.L.Z., Z.W.L. J.X., and T.H.H. performed the experiments. Y.Y.X. analysed the data and drafted the manuscript. T.H.H. prepared the Figures. T.A. helped in yeast screening, discussion during analysis and reviewing of the manuscript.

Figure S1 Analysis of OsATL15 nucleotide sequence and its conserved domain to deduce its amino acid sequence.

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Figure S2 Generation of osatl15 knockout mutants in rice.

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Figure S3 Thiamethoxam (THX) uptake kinetics in OsATL15 rice genotype.

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Tables S1 Percentage change in THX content in OsATL15 transgenic lines compared with the WT over a time course of chemical exposure.

Table S2 Percentage change in THX content in OsATL15 transgenic lines, compared with the WT under different THX concentration treatments.

Table S3 Mortality of third‐instar nymphs of brown planthopper (BPH) on rice treated with THX.

Table S4 Pesticides analysed by UPLC–MS/MS using ESI and EI ionization, retention time, and cone voltage and collision energy for each transition monitored in the SRM mode.

Table S5 Primers used in the present study.

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