Chemicals and reagents
The following chemical reagents were procured from commercial suppliers: BA, benzylbenzoate, benzylsalicylate and phenethyl phenylacetate from Shanghai Macklin Biochemical Technology. SGE (ZB-240319) from Shanghai ZZBIO; and deuterium-labelled BA (HY-N0216S) and benzylbenzoate (HY-B0935S1) were purchased from MedChemExpress. SAG was provided by C. Song.
For molecular and biochemical blockays, the following kits were used: RNA extraction kit (Easy-Do, DR0409050), reverse transcription kit (TransGen Biotech, AE311-02), qPCR kit (Vazyme Biotech, Q711-02), Clonexpress II One Step Cloning Kit (Vazyme Biotech, C11202). Enzyme activity blockays were performed using Alcohol Dehydrogenase Activity Assay Kit (Beyotime Biotechnology, S0241S), Catalase (CAT) Assay Kit (Beijing Solarbio Science and Technology, BC0200), Protein Marker (Yeasen Biotechnology, 26616ES72), and the Bradford Protein Assay Kit (Coolaber, SK1060). For cloning and protein expression, the following competent cells were used: DH5α (Tsingke, TSC-C01), Rosetta 2 Competent cells (Weidi, EC1014) and GV3101 (Angyubio G6039).
Plant materials and growth conditions
The rice (O. sativa) mutants cnl, bebt, bbh and bse are on the background of the japonica cultivar ZH11 (zhonghua11), and the rice mutants te1 te2 and ics1 are on the background of the japonica cultivar NIP (Nipponbare). The cnl mutants were generated in our previous study28, and bebt, bbh and bse mutants are CRISPR–Cas9 mutants obtained from Biogle Genetech (https://www.biogle.cn). The rice te1 te2 mutants were generated using CRISPR–Cas9 and ics1 mutants were obtained from J.-L. Qiu18. Rice seeds were collected at maturity and dried at 42 °C for 4 days to break seed dormancy and fully filled seeds were selected for experiments. In the greenhouse, except for the rice plants used for pathogen treatment experiments, all rice plants were grown under a 14 h:10 h light:dark photoperiod, 30 °C/28 °C day/night temperature, and 85% humidity. Rice plants used for pathogen treatment experiments were grown in trays filled with field soil under the 14:h light:10 h dark photoperiod, 28 °C/26 °C day/night temperature, and 60% humidity. The Arabidopsis thaliana mutants Atbebt-1 (SALK_030095), Atbebt-2 (SALK_089260), Atbse (SALK_205911) and Atbbh (SALK_039417), obtained from Arabidopsis Biological Resource Center (ABRC), were grown in a growth chamber under 16 h:8 h light:dark and 22 °C/20 °C day/night cycles. Atics1 (sid2-1) was reported in a previous study9. The tomato (S. lycopersiblock L. cultivar M82) seedlings were grown in a growth chamber under 16 h:8 h light:dark photoperiod, 24 °C/22 °C day/night temperature, and 60% humidity. The wheat (T. aestivum L.) seedlings were grown in a growth chamber under 16 h:8 h light:dark photoperiod, 23 °C/21 °C day/night temperature, and 60% humidity. The cotton (G. hirsutum L. cultivar TM-1) seedlings were grown in a growth chamber under a 16 h:8 h, light:dark photoperiod, 25 °C/23 °C day/night temperature, and 40% humidity. N. benthamiana plants were grown in a growth chamber at 22 °C with 16-h:8-h light:dark photoperiod, 22 °C/20 °C day/night temperature, and 60% humidity.
RNA extraction and vector construction
Total RNA was extracted using the Rice RNA Rapid Extraction Kit (Easy-Do, DR0409050) and was reverse-transcribed using the reverse transcription kit (TransGen Biotech, AE311-02), after which cDNA was used for gene cloning and RT–qPCR. qPCR was performed according to instructions from the qPCR kit (Vazyme Biotech, Q713-02) using the Light Cycler Instrument 480 II (Roche). Vectors were constructed by homologous recombination using Clonexpress II One Step Cloning Kit (Vazyme Biotech, C11202). Primers for gene cloning are listed in Supplementary Table 1, RT–qPCR, semi-quantitative PCR, DNA-based qPCR, rice mutant characterization, and restriction enzyme sites and plasmids used for subcellular localization, transient expression, recombinant protein expression and VIGS are listed in Supplementary Table 2.
Transient protein expression in plant cells
Vectors used for Agrobacterium-mediated transient expression in N. benthamiana were transformed into A. tumefaciens strain GV3101, using the freeze-thaw method, and spread onto LB agar plates (50 μg ml−1 kanamycin and 30 μg ml−1 rifampicin) to grow at 28 °C for 2 days. The positive transformants were inoculated into 2 ml of liquid LB cultures (50 μg ml−1 kanamycin and 30 μg ml−1 rifampicin) with shaking for 16 h at 30 °C. GV3101 were collected by centrifugation at 6,000g for 5 min and resuspended in 10 mM MgCl2 solution containing 200 μM acetosyringone and later injected into the abaxial surface of 4- to 6-week-old N. benthamiana leaves with a needleless syringe, after which the infiltrated leaves were collected for blockysis within 2–3 days51. For protoplast transformation, polyethylene glycol-mediated transfection of isolated rice protoplasts were performed as described previously52.
Subcellular localization
To determine protein subcellular localization, infiltrated N. benthamiana leaves were collected 48 h post-infiltration for observation, and the transformed rice protoplasts were incubated in darkness for 12–16 h before observation. The endoplasmic reticulum marker ER-rk, which was created by fusing the signal peptide of AtWAK2 (Arabidopsis wall-blockociated kinase2) at the N-terminus of the mCherry protein and the endoplasmic reticulum retention signal His-Asp-Glu-Leu at its C-terminus, was obtained from ABRC (CD3-959). A Fluoview FV3000 confocal laser-scanning microscope (Olympus) was used for image capturing in epidermal cells, where CFP was excited with 414 nm lasers and detected at 460–500 nm, YFP was excited with 475 nm lasers and detected at 505–550 nm, and mCherry was excited with 587 nm lasers and detected at 600–650 nm.
Expression of recombinant proteins in E. coli
Protein expression in E. coli was conducted using a previously reported method28. Constructs for the His-tagged recombinant proteins were first transformed into E. coli Rosetta (Weidi, EC1014), cultured in 1 ml LB medium overnight at 37 °C with 220 rpm of shaking, and then added to 100 ml LB medium for incubation for 2–3 h at 37 °C with 220 rpm of shaking. After the bacterial OD600 reached 0.4–0.5, 0.5 mM isopropyl β-d-1-thiogalactopyranoside was added, and the mixture was incubated overnight at 18 °C with shaking at 120 rpm. The bacteria were subsequently collected by centrifugation and resuspended in 30 ml lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, pH 7.4), lysed by sonication followed by centrifugation at 12,000 rpm for 1 h. The supernatant was collected, from which the recombinant proteins were purified using Ni-NTA beads (Smart lifesciences, SA003025). Protein concentration was determined using the Bradford Protein Assay Kit (Coolaber, SK1060) according to the manufacturer’s instructions.
Feeding of BA, benzylbenzoate and benzylsalicylate to rice seedlings
BA, benzylbenzoate, benzylsalicylate, deuterium-labelled BA and deuterium-labelled benzylbenzoate were dissolved in isopropanol to prepare a 200 mM stock solution, which was then diluted to a working concentration of 200 μM using water containing 0.01% Silwet-L77. Rice seeds were immersed in the solution and grown in a greenhouse under a 14 h:10 h light:dark photoperiod and 30 °C/28 °C day/night temperature. After five days of growth, the metabolites in the emerged leaves were extracted and blockysed.
Enzymatic characterization of OsTE
The thioesterase activity of OsTE was blockayed according to a previous report41, with minor modifications. In brief, 150 μl buffer containing 50 mM KH2PO4 at pH 7.0 with the addition of 500 μM benzoyl-CoA or cinnamoyl-CoA were mixed with 1 μg of purified recombinant proteins in 10 μl solution, and incubated at 28 °C. After 60 min, the reaction was terminated by adding 80 μl 10% trichloroacetic acid aqueous solution, and was sent for blockysis by HPLC.
For enzyme kinetic blockysis, 1 μg recombinant protein was added to 150 μl of the blockay buffer containing varied concentrations of substrate (3.9 μM to 500 μM). After 1 min at 28 °C, the reaction was terminated, and the production of BA or cinnamic acid was blockysed by ultrahigh performance liquid chromatography (UHPLC).
Enzymatic characterization of OsBEBT
The acyltransferase activity of OsBEBT was blockayed according to a previous report36, with minor modifications. For reactions, each substrate (500 μM) was incubated with 1 μg purified recombinant protein in a total volume of 150 μl of the reaction buffer that consisted of 50 mM Tris-HCl and 300 mM NaCl (pH 7.0). After incubating at 28 °C for 60 min, the reaction was terminated by adding 80 μl 10% trichloroacetic acid aqueous solution, followed by HPLC blockysis.
For kinetic blockysis, 1 μg of the recombinant protein was added to 150 μl of the blockay buffer containing a saturated concentration (500 μM) of benzyl alcohol, isovanillyl alcohol, cinnamyl alcohol, vanillic alcohol, or ethanol, respectively, and varied concentrations of BA-CoA (3.9 μM to 500 μM). After 1 min at 28 °C, 80 μl 10% trichloroacetic acid aqueous solution was added to terminate the reaction. Reaction products were detected and quantified using UHPLC. Benzylbenzoate, ethyl benzoate and benzylcinnamate were quantified using standard curves generated from blockytical standards. For other esters, quantification was based on monitoring the consumption of the corresponding alcohol.
Enzymatic characterization of OsBBH
Detached leaves were incubated with substrates for catalysis. Analysis was performed according to a previous report53. In brief, A. tumefaciens strain GV3101 harbouring pEARLY100:35S-OsBBH and pEARLY100:35S-mVenus were infiltrated into different regions of the same N. benthamiana leaf. After 72 h, infiltrated leaves were collected for RNA extraction and subsequent expression level blockysis. For metabolite blockysis, approximately 0.02 g of the infiltrated leaves were collected and incubated in benzylbenzoate solution (200 μM benzylbenzoate dissolved in 0.01% Silwet-77) for 16 h. Then, the leaves were washed with de-ionized water, ground in liquid nitrogen, and homogenized in 0.5 ml methanol, with the addition of 173 ng phenethyl phenylacetate as internal standard. The supernatant was dried with nitrogen and dissolved in 150 μl 70% methanol, followed by extraction with 150 μl hexane. The hexane phase was collected for GC–MS blockysis. All reactions in each batch of the experiments were conducted with five biologically independent samples.
Microsomal protein preparation and in vitro enzyme blockays were conducted according to previous studies54,55. In brief, pEARLY100:35S-OsBBH was transiently expressed in N. benthamiana leaves. Then, the microsomal proteins were prepared from N. benthamiana leaves and used for blockysis. Leaves expressing pEARLY100:35S-mVenus were used as control. Approximately 0.5 g of N. benthamiana leaves were ground into fine powder in liquid nitrogen and then resuspended in 30 ml of microsome extraction buffer (50 mM tris-HCl, 2 mM EDTA, 2 mM 2-mercaptoethanol and 0.4 M sucrose, pH 7.4). The homogenate was incubated on ice for 20 min, followed by filtering through 2 layers of Miracloth (Millipore), and centrifuged at 10,000g for 10 min. The supernatant was collected for further centrifuged at 100,000g for 1 h. The resulting pellet was dissolved in 100 μl resuspension buffer (100 mM HEPES, pH 7.4). Microsome protein concentration was measured using a Bradford kit. For the BBH catalytic activity blockay, 5–10 μg of microsomal proteins were added into 100 μl reaction buffer containing 50 mM HEPES (pH 7.4), 0.005% Silwet, 200 μM benzylbenzoate, and 2 mM NADPH. After incubation in 28 °C for 1 h, 50 μl of internal standard solution containing 0.005% Silwet and 0.54 μg of phenethyl phenylacetate as internal standard was added and the mixture was extracted with 200 μl of ethyl acetate. The ethyl acetate phases were collected and blockysed by GC–MS.
Enzymatic characterization of OsBSE
The esterase activity of OsBSE was blockysed according to a previous report43, with minor modifications. In brief, 150 μl buffer containing 50 mM Tris-HCl at pH 7.0 and 200 μM benzylsalicylate were mixed with 1 μg of purified proteins in 10 μl solution to start the enzymatic reaction. After 60 min at 28 °C, 80 μl 10% trichloroacetic acid aqueous solution was added to terminate the reaction. The solution was sent for blockysis by HPLC.
For enzyme kinetic blockysis, 1 μg recombinant protein were added to 150 μl blockay buffer containing varied concentrations of benzylsalicylate, benzylbenzoate, or cinnamylbenzoate (3.9 μM to 500 μM). After 10 min at 28 °C, the reaction was terminated as mentioned. The content of SA or BA produced was blockysed by UHPLC.
Extraction and enzymatic profiling of cytosolic and non-cytosolic protein fractions from rice leaves
Cytosolic and non-cytosolic protein fractions were extracted from four-week-old rice leaves using a modified protocol based on a previous report56. In brief, 8 g of rice leaves were homogenized in ice-cold extraction buffer (50 ml, 0.5 M sorbitol, 20 mM HEPES, 10 mM KCl, 5 mM 2-mercaptoethanol, pH 7.0). The homogenate was filtered through two layers of Miracloth (Merck millipore) and centrifuged at 10,000g for 60 min. The supernatant was carefully collected as cytosolic fraction, while the precipitation was resuspended in 2 ml 50 mM Tris-HCl to obtain the non-cytosolic fraction. Protein concentrations in both fractions were determined using the Bradford kit (Coolaber, SK1060). For enzymatic blockysis of BSE, the fractions containing 5 μg proteins were added into 150 μl buffer containing 500 μM benzylsalicylate and incubated at 28 °C for 5 min. The reaction products were blockysed by UHPLC. The cytosolic fraction was verified by measuring alcohol dehydrogenase (ADH) activity, while the non-cytosolic fraction was confirmed by measuring catalase (CAT) activity and chlorophyll content56. The ADH and CAT activities were measured using the ADH Assay Kit (Beyotime Biotechnology, S0241S) and the CAT Assay Kit (Beijing Solarbio Science & Technology, BC0200) following manufactures’ instructions. Chlorophyll contents were determined by measuring the absorbance at 649 nm and 665 nm. Concentrations of chlorophylls a and b were calculated using extinction coefficients as previously described57. All absorbances were detected by a microplate reader (BioTek, Synergy H1, Agilent).
Extraction and immunoblotting of cytosolic and non-cytosolic protein fractions from rice protoplasts
Rice protoplasts expressing BSE–YFP and ER-mCherry were collected and homogenized in ice-cold extraction buffer, and the homogenate was then centrifuged at 10,000g for 60 min at 4 °C. The supernatant was carefully collected as the cytosolic fraction, while the pellet was resuspended in 50 mM Tris-HCl (pH 7.4) to obtain the non-cytosolic fraction. Protein concentrations in both fractions were determined using the Bradford kit (Coolaber, SK1060). Proteins were separated by SDS–PAGE and detected via immunoblotting using the following primary antibodies: anti-Histone H3 (1:5,000; ab1791, Abcam), anti-GAPDH (1:5,000; HRP-60004, Proteintech), anti-YFP/GFP (1:4,000; E-A02020, Abbkine), and anti-mCherry/RFP (1:5,000; ab65856, Abcam). Immunoblots were developed using the Super ECL Star chemiluminescent substrate (US Everbright). Images from immunoblotting were collected with Tanon-5200 Chemiluminescent Imaging System (Tanon Science and Technology).
Virus-induced gene silencing
VIGS in S. lycopersiblock was conducted using the bipartite tobacco rattle virus (TRV) vectors, pTRV1 and pTRV2, according to a previous report58. Putative orthologues of OsBEBT, OsBBH or OsBSE were selected as VIGS target genes. cDNA fragments (200–300 bp) of SlBEBT (Solyc07g049660), SlBBH (Solyc03g122350) and SlBSE (Solyc02g069800), amplified using primers listed in Supplementary Table 1, were inserted into the pTRV2 vector and transformed into A. tumefaciens strain GV3101. The empty pTRV2 vector was used as a negative control, while pTRV2 containing SlPDS (encoding phytoene desaturase) fragments was used as a positive control. A. tumefaciens GV3101 harbouring pTRV1 or pTRV2 were cultivated, resuspended in transformation buffer, and mixed in equal amount. Two-day-old tomato seedlings were soaked in the Agrobacterium suspension and subjected to vacuum infiltration.
VIGS of TaBEBT, TaBBH, and TaBSE in wheat seedlings was performed using barley stripe mosaic virus (BSMV) system as described59,60. BSMV contains three positive-sense, single-stranded RNA segments (α, β and γ). The γ segment can be modified at the cDNA level to incorporate fragments of target genes for VIGS. Putative orthologous genes of OsBEBT, OsBBH, or OsBSE were selected as VIGS target genes. In brief, the highly conserved cDNA sequence fragments (200–300 bp) of TaBEBT homologues (TraesCS1D03G0425100, TraesCS1D03G0424200, TraesCS1B03G0562000 and TraesCS1A03G0468700), TaBBH homologues (TraesCS5D03G0509500, TraesCS5D03G0509300, TraesCS5B03G0555800, TraesCS5B03G0555700, TraesCS5A03G0546200 and TraesCS5A03G0546100) and TaBSE homologues (TraesCS1D03G0617800, TraesCS1D03G0617300, TraesCS1B03G0749400, TraesCS1B03G0747800, TraesCS1A03G0660500 and TraesCS1A03G0659500) were inserted into the RNAγ cDNA strand to prepare cDNA clones of BSMV:TaBEBT, BSMV:TaBBH or BSMV:TaBSE, respectively (primers and insertion sites are listed in Supplementary Table 1). The vector containing the TaPDS cDNA fragment was used as a positive control and the empty vector as a negative control. The RNAα, RNAβ and RNAγ-derived clones were linearized and the in vitro transcription was carried out in accordance with the Ribo MAXTM Large Scale RNA Production System-T7 kit and the Ribom7G Cap Analog kit (Promega, P1712). Then, the synthesized RNA transcripts of RNAα, RNAβ and RNAγ were mixed in equal molar ratios and diluted for inoculation onto wheat leaves at the two-leaf stage.
VIGS of GhBEBT, GhBBH and GhBSE in cotton seedlings was performed as described61. Putative orthologous genes of OsBEBT, OsBBH or OsBSE were selected as VIGS target genes. The highly conserved cDNA sequence fragments (200–300 bp) of GhBEBT (Gohir.D13G163100.1 and Gohir.A13G158400.1), GhBBH (Gohir.D11G140901.1 and Gohir.A11G134700.1), GhBSE (Gohir.D06G184200.1 and Gohir.A06G174100.1), amplified using primers listed in Supplementary Table 1, were inserted into the pTRV2 vector. An equal volume of A. tumefaciens GV3101 harbouring pTRV1 and different pTRV2 vectors were mixed and infiltrated into the cotyledons of two-week-old cotton seedlings. The empty pTRV2 vector served as a negative control, while pTRV2 containing a fragment of the cloroplastos alterados (CLA) gene was used as a positive control.
Putative orthologous genes of OsBEBT, OsBBH or OsBSE from different plants were obtained through reciprocal protein BLAST. Silencing efficiency in the above VIGS experiments was confirmed in the positive control group (plants with silenced SlPDS, TaPDS or GhCLA) by the appearance of an albino phenotype, which also indicated the appropriate time for collection or further pathogen infection experiments in the target gene-silenced groups.
Pathogen treatment
M. oryzae were cultivated in a growth chamber at 28 °C with a 16 h:8 h light:dark photoperiod for 10 days. Spores were collected and resuspended in 0.01% gelatin to make the concentration of 5 × 106 per ml. For fungal biomblock evaluation, young leaves from 4-leaf-stage rice were cut into evenly sized leaf segments and slightly scratched to facilitate inoculation. A 10 μl aliquot of spore suspension were inoculated on each wound, and the leaf segments were incubated at 28 °C. Seven days post-inoculation, disease symptoms were measured using ImageJ 1.42q software (https://imagej.nih.gov/ij/), and the relative fungal biomblock was calculated using DNA-based qPCR by measuring the level of the M. oryzae transposable element MoPot2 relative to that of OsUBIQUITIN62.
The bacterial strain Pseudomonas syringae DC3000 was cultured in Luria–Marine liquid overnight. The following day, the bacteria were collected, washed with sterile water, and resuspended to reach the final concentration of 1 × 106 per ml. The bacteria were then inoculated into Arabidopsis leaves using a syringe, and the inoculated plants were kept under ambient humidity for approximately 1 h to allow for water evaporation before being transferred to a growth chamber5. Two days post-inoculation, Arabidopsis leaves were collected for PCR with reverse transcription (RT–PCR) and metabolite blockysis.
For S. lycopersiblock inoculation, freshly prepared DC3000 from the nutrient yeast glycerol agar (NYGA) plate containing 50 μg ml−1 rifampicin was resuspended in 10 mM MgCl2 and 0.02% Silwet-77. OD600 was adjusted to 0.01 before the solution was sprayed on tomato leaves. Leaves were collected for further blockysis 24 h after inoculation.
F. graminearum for wheat pathogen infection was cultured on potato dextrose agar and grown at 28°C for 5 days before being inoculated into mung bean soup medium to induce spore production. After 7 days of growth in a shaking incubator at 28 °C, the conidia suspension of F. graminearum (1 × 105 per ml) was evenly sprayed on wheat leaves. Sterile distilled water was inoculated in the same way as the control. Two days after inoculation, the leaves were collected for RT–PCR and metabolite blockysis.
Verticillium dahlia V991 for cotton pathogen infection was cultivated on potato dextrose agar for four days, and the spores were collected and grown in Czapek’s liquid medium. Cotton seedlings were inoculated with spore suspensions (8 × 106 per ml) through injured roots. The leaves were collected three days after inoculation61.
Plant metabolite extraction
Extraction of plant SA and BA was performed according to a previous report28 with minor modifications. In brief, ~100 mg of plant leaves was ground and added to 0.5 ml of ethyl acetate containing 2.5 ng deuterium-labelled SA (SA-d4, GLPBIO, GC49480), followed by vigorous shaking for 10 min. The samples were subsequently centrifuged for 10 min at 4 °C before the supernatant was collected. Then, 0.5 ml ethyl acetate containing 2.5 ng SA-d4 was added to the precipitate, followed by vigorous shaking for 10 min, and subsequently centrifuged for 10 min at 4 °C. The supernatants from the above two centrifugations were combined, vacuum evaporated, re-dissolved in 70% methanol, and transferred to an injection vial to be blockysed by LC–MS/MS.
Extraction of plant SAG and SGE was performed according to a previous report63 with minor modifications. In brief, around 100 mg of plant leaves was ground and added to 1 ml of 75% methanol containing 5 ng deuterium-labelled SA. The mixture was incubated at 4 °C for 12 h, followed by centrifugation at 12,000g for 10 min at 4 °C. The resulting supernatant was carefully collected and subjected to LC–MS/MS blockysis for quantification.
Extraction of rice benzylbenzoate and benzylsalicylate was conducted as follows. Approximately 5 g of rice leaves were ground with nitrogen and added to 20 ml ethyl acetate containing 5 ng SA-d4, followed by sonification for 30 min. The samples were subsequently centrifuged for 10 min, and the supernatant was collected and vacuum vapoured. The concentrate was re-dissolved in 3 ml 70% methanol and extracted by 2.8 ml hexane. The hexane phase was collected and concentrated by nitrogen gas, and re-dissolved in 80 μl methanol for LC–MS/MS blockysis.
Extraction of benzyl-CoA was performed as described64. In brief, 200 mg of rice leaves were ground in liquid nitrogen, and resuspended with 300 μl of 10% TCA solution. Samples were centrifuged for 10 min at 4 °C, after which this extraction was repeated. Combined supernatants were mixed with an equal volume of 8% ammonium acetate solution, then centrifuged to precipitate residual impurities. Solid-phase extraction (SPE) cartridges (1 cc, 50 mg, C18 Sep Pak Vac, Waters) were preconditioned with 2 ml methanol, 2 ml ultrapure water, and 2 ml 4% ammonium acetate in 5% TCA. Samples were loaded to the columns and washed with 2 ml 4% ammonium acetate. Columns were flushed with air and eluted with 200 μl of 80% isopropanol and 20% ethyl acetate. The elution was collected for LC–MS/MS blockysis.
HPLC blockysis
For initial enzyme activity test, the substrates and products of OsBEBT, OsBSE, and OsTE were detected by HPLC (Shimadzu LC-20A) equipped with a Photo-diode array at a wavelength of 210 nm. Samples were separated with a Welch Ultimate XB-C18 column (250 × 4.6 mm, 5 μm particle size). For each run, 20 μl of each sample were injected into a column at 30 °C. The mobile phase consisted of solvent A (0.1% phosphoric acid in distilled water) and solvent B (acetonitrile) with a flow rate of 1 ml min−1. A gradient elution program was used as follows: 0–20 min, 80% to 20% A; and 20–25 min, 20% to 0% A.
UHPLC blockysis
For kinetics blockysis, the enzymatic products of OsBEBT, OsBSE and OsTE were quantified by UHPLC (Vanquish Flex, Thermo Fisher Scientific) equipped with a variable wavelength detector or a fluorescence detector. Samples were separated with a Sepax HP-C18 column (2.1 mm × 150 mm, 3 μm particle size) maintained at 40 °C. For each run, 10 μl of each sample were injected into a column at 25 °C. The mobile phase consisted of solvent A (HPLC-grade methanol) and solvent B (distilled H2O with 0.1% formic acid) with a flow rate of 0.5 ml min−1. A gradient elution program was used as follows: 0–1 min, 95% B; 1–8 min, 95% to 50% B; 8–10 min, 50% to 0% B; 10–15 min, 0% B; 15–15.5 min, 0% to 95% B; and 15.5–19 min, 95% B. SA was detected by the fluorescence detector, with the excitation wavelength of 295 nm and emission wavelength of 400 nm. The wavelengths for the detection of benzylbenzoate and other chemicals were 194 nm and 210 nm. Quantification of the detected products was performed using standard curves for BA, benzylbenzoate, benzylsalicylate, cinnamic acid and SA based on UV or visible light absorbance.
LC–MS/MS with MRM blockysis
LC–MS/MS blockysis was performed utilizing an ExionLC HPLC system coupled with an AB SCIEX QTRAP 6500plus mblock spectrometer. Aliquots of 2 µl were injected into an Acquity BEH C18 column (2.1 × 50 mm). Chromatographic separation was achieved with a flow rate of 0.4 ml min−1, using 0.1% formic acid in water (solvent A) and acetonitrile (solvent B). Liquid chromatography separation was performed using an 8-min linear elution gradient: 0 min, 5% B; 1.5 min, 20% B; 3.5 min, 50% B; 5 min, 90% B; 5–6.5 min, 90% B; 6.6 min, 5% B; and 6.6–8 min, 5% B. BA, SA, SAG, SGE and deuterium-labelled SA (SA-d4, GLPBIO, GC49480) were blockysed in negative ion-mode electrospray ionization (ESI−), while benzylbenzoate, benzylsalicylate, deuterium-labelled benzylbenzoate and deuterium-labelled benzylsalicylate was blockysed in positive ion-mode (ESI+). MRM mode quantification settings were: curtain gas at 35 psi; ion spray voltage at −4.5 kV for ESI− and +5.5 kV for ESI+; ion source temperature at 500 °C; ion source gases 1 and 2 at 60 and 40 psi, respectively. MRM transitions were set at 121.0/77.0 for BA, 137.0/93.0 for SA, 141.0/97.0 for SA-d4, 299.2/137.2 for SAG, 299.2/150.9 for SGE, 229.1/91.1 for benzylsalicylate, and 213.0/91.0 for benzylbenzoate, 240.1/98.1 for deuterium-labelled benzylsalicylate, 223.1/96.1 for deuterium-labelled benzylbenzoate. Declustering potential and collision energy values were −30 V/−17 V for BA, −35 V/−21 V for SA, −60 V/−16 V for SAG, −60 V/−30 V for SGE, −35 V/−20 V for SA-d4, 50 V/33 V for benzylsalicylate, and 50 V/26 V for benzylbenzoate, 50 V/33 V for deuterium-labelled benzylsalicylate, 50 V/26 V for deuterium-labelled benzylbenzoate. A 20 ms dwell time was applied. Quantification was performed using standard curves established with blockytical standards and a 5 ng ml−1 SA-d4 internal standard.
For benzoyl-CoA detection, the ExionLC HPLC system coupled with an AB Sciex QTrap 6500plus mblock spectrometer was used. Aliquots of 2 µl were injected into an Acquity BEH Amide column (2.1 × 50 mm). Chromatographic separation was achieved with a flow rate of 0.4 ml min−1, with 10 mM ammonium acetate and 0.05% ammonium hydroxide in water (solvent A) and 10 mM ammonium acetate and 0.05% ammonium hydroxide in 90% acetonitrile (solvent B). An 8-min linear gradient was employed for elution. The gradient program was as follows: 0–1 min, 95% B; 1–2.5 min, 95% to 55% B; 2.5–5 min, 55% B; 5–5.1 min, 55% to 95% B; and 5.1–8 min, 95% B. Benzoyl-CoA and deuterium-labelled benzoyl-CoA were blockysed in positive ion-mode (ESI+). MRM mode quantification settings were: curtain gas at 40 psi; ion spray voltage at +4.5 kV for ESI+; ion source temperature at 500 °C; ion source gases 1 and 2 at 55 and 50 psi, respectively. MRM transitions were set at 872.2/365.0 for benzoyl-CoA, 877.2/370.0 for deuterium-labelled benzoyl-CoA. Declustering potential and collision energy values were 100 V/47 V for benzoyl-CoA, and 100 V/47 V for deuterium-labelled benzoyl-CoA. A 100 ms dwell time was applied with quantification using a standard curve established with blockytical standards.
GC–MS blockysis
One microlitre sample was auto-injected onto Agilent GC 8890 equipped with an HP-5MS capillary column and an Agilent 5977B MS detector. Temperature of the gas chromatography oven was programmed as follows: initial hold at 45 °C for 2 min, followed by sequential increases of 10 °C/min to 90 °C, 3 °C/min to 105 °C, and 10 °C/min to 280 °C, with a final hold at 280 °C for 7 min. Mblock spectra were acquired in scan mode (35–300 m/z) for qualitative blockysis or selected ion monitoring (SIM) mode (m/z = 91, 104, 212, 228) for quantitative blockysis. The peak area of the EIC at m/z = 228 was used to quantify benzylsalicylate, while the peak area of EIC at m/z = 104 was used to quantify phenethyl phenylacetate. The response factor of benzylsalicylate relative to phenethyl phenylacetate was determined using standard curves generated from authentic standards and applied for the quantification of benzylsalicylate.
Phylogenetic blockysis
Rice protein sequences were downloaded from the Rice Annotation Project Database (RAP-DB)65 and those of N. tabablock were retrieved from NCBI (GCF_000715135.166). Homologues of each gene were identified using DIAMOND67 v2.0.14, with the query gene and an e-value threshold of 10−10. The identified sequences were aligned using MAFFT68 v7.490, and poorly aligned regions were trimmed using TrimAl69 v1.4. A maximum likelihood phylogenetic tree was constructed using IQ-TREE70 v2.3.0 with the “LG + F + G4” model and 1,000 ultrafast bootstrap replicates71.
Gene co-expression blockysis
ATTED-II (https://atted.jp), a gene co-expression resource based on publicly available RNA-sequencing and microarray data72, was used to identify genes co-expressed with OsCNL1 (Entrez Gene ID: 4331500). OsCNL1 was queried in the ATTED-II database (https://atted.jp/top_search/#CoExSearch), and the top 2,000 co-expressed genes were ranked based on their z-scores, which indicate co-expression strength. These genes were visualized around OsCNL1 using the Cytoscape software (v.3.7.2), with their distance to OsCNL1 reflecting the strength of co-expression.
Statistical blockysis and reproducibility
Statistical significance was blockessed by two-tailed Student’s t-tests (for pairwise comparisons) or one-way ANOVA with Tukey’s HSD post hoc test. When the blockumption of equal variances was violated (as determined by Bartlett’s test), Welch’s ANOVA with Games–Howell post hoc tests was applied. All statistical blockyses were performed using GraphPad Prism 8.0 (https://www.graphpad.com/). Kinetic parameters were derived by fitting data to the Michaelis–Menten equation using nonlinear regression in the same software. Specific statistical tests and sample sizes are indicated in the respective figure legends. Sample sizes were not predetermined using statisitical methods. Most experiments were performed with at least three biological replicates, which is the minimum number required for robust statistical blockysis of metabolite data and sufficient to blockess experimental variability. For specilized experiments, such as VIGS and tobacco transient expression, three to six replicates were used to ensure the reliability of the results. Sample allocation was random. Blinding was not used in this study.
For immunoblot blockysis, fluorescence microscope blockysis and semi-quantitative RT–PCR blockysis, representative images were shown and each experiment was independently repeated at least three times with similar results to ensure reproducibility.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
