No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.
Chemical compounds and standards
Tropine, (S)-hyoscyamine hydrobromide and (S)-scopolamine hydrobromide were purchased from Santa Cruz Biotechnology. (R)-Littorine hydrochloride was purchased from Toronto Research Chemicals. All other chemicals were purchased from Sigma.
DNA oligonucleotides used in this study were synthesized by the Stanford Protein and Nucleic Acid Facility and are listed in Supplementary Data 1. Genes encoding biosynthetic enzymes used in this study are listed by source and accession number in Supplementary Table 2; for HDH genes newly identified in this work, full amino acid sequences are given in Supplementary Table 1. Endogenous yeast genes were amplified from Saccharomyces cerevisiae CEN.PK2-1D51 genomic DNA via colony PCR52. Gene sequences encoding heterologous enzymes were codon-optimized for expression in S. cerevisiae using GeneArt GeneOptimizer software (Thermo Fisher Scientific) and synthesized as double-stranded gene fragments (Twist Bioscience). Plasmids used in this study are listed in Supplementary Data 2. Three types of plasmids were used in this work: yeast expression plasmids, yeast integration plasmids, and Agrobacterium tumefasciens binary vectors.
Yeast expression plasmids harboured a gene of interest flanked by a constitutive promoter and terminator, an auxotrophic selection marker, and either a low-copy CEN6/ARS4 or a high-copy 2μ yeast origin of replication. These plasmids were constructed by addition of 5′ and 3′ restriction sites to genes of interest using PCR, restriction digestion of PCR amplicons or synthesized gene fragments, and ligation of digested inserts into similarly digested vectors pAG414GPD-ccdB, pAG415GPD-ccdB, pAG416GPD-ccdB, pAG424GPD-ccdB, pAG425GPD-ccdB or pAG426GPD-ccdB53 using T4 DNA ligase (New England Biolabs, NEB). Yeast expression plasmids expressing fusions of multiple proteins or enzymes were prepared by PCR amplification of each gene of interest with 15–25 bp of overlap to adjacent fragments, assembly of fragments into single inserts with 5′ and 3′ restriction sites using overlap-extension PCR, and ligation cloning into digested vectors as described.
Yeast integration plasmids comprised a gene of interest flanked by a constitutive promoter and terminator, but lacked a selection marker and origin of replication for yeast expression. These plasmids were constructed by PCR linearization of the empty holding vectors pCS2656, pCS2657, pCS2658, pCS2661 or pCS2663 using primers complementary to the 3′ and 5′ ends of the promoter and terminator, respectively. Genes intended for yeast genomic integration were PCR-amplified to append 5′ and 3′ overhangs with 35–40 bp of homology to the termini of the linearized holding vectors and then assembled using Gibson assembly.
For transient expression of littorine synthase variants in Nicotiana benthamiana, A. tumefasciens binary vectors contained a transfer-DNA (T-DNA) region comprising a gene of interest flanked by the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter/Cowpea Mosaic Virus (CPMV) 5′UTR and a nopaline synthase terminator, as well as an analogous expression cassette for the p19 RNAi-suppressor protein. These plasmids were constructed via addition of 5′ AgeI and 3′ XhoI restriction sites to a gene of interest via PCR, followed by digestion and ligation into the pEAQ-HT binary vector pCS335254.
All PCR amplification was performed using Q5 DNA polymerase (NEB) and linear DNA was purified using the DNA Clean and Concentrator-5 kit (Zymo Research). Assembled plasmids were transformed into chemically competent E. coli (TOP10, Thermo Fisher Scientific) via heat-shock and propagated with selection in Luria–Bertani (LB) broth or on LB-agar plates with either carbenicillin (100 μg ml−1) or kanamycin (50 μg ml−1) selection. E. coli plasmid DNA was isolated by alkaline lysis from overnight cultures grown at 37 °C and 250 rpm in selective LB media using Econospin columns (Epoch Life Science) according to the manufacturer’s protocol. Plasmid sequences were verified by Sanger sequencing (Quintara Biosciences).
Yeast strain construction
Yeast strains used in this study (Supplementary Table 3) were derived from our previously reported tropine-producing strain CSY125114, which is in turn derived from the parental strain CEN.PK2-1D51. Strains were grown non-selectively in yeast-peptone media supplemented with 2% w/v dextrose (YPD media), yeast nitrogen base (YNB) defined media (Becton, Dickinson and Company, BD) supplemented with synthetic complete amino acid mixture (YNB-SC; Clontech) and 2% (w/v) dextrose, or on agar plates of the aforementioned media. Strains transformed with plasmids bearing auxotrophic selection markers (URA3, TRP1 and/or LEU2) were grown selectively in YNB media supplemented with 2% w/v dextrose and the appropriate dropout solution (YNB-DO; Clontech) or on YNB-DO agar plates.
Yeast genomic modifications were performed using the CRISPRm method55. CRISPRm plasmids expressing Streptococcus pyogenes Cas9 and a single guide RNA (sgRNA) targeting a genomic locus were constructed by assembly PCR and Gibson assembly of DNA fragments encoding SpCas9 (pCS3410), tRNA promoter and HDV ribozyme (pCS3411), a 20-nucleotide guide RNA sequence oligonucleotide, and tracrRNA and terminator (pCS3414) (Supplementary Data 2). For gene insertions, integration fragments comprising one or more genes of interest flanked by unique promoters and terminators were PCR-amplified from yeast integration plasmids using Q5 DNA polymerase (NEB) with flanking 40 bp microhomology regions to adjacent fragments and/or to the yeast genome at the integration site (Extended Data Fig. 1, Supplementary Data 1). For gene disruptions, integration fragments comprised 6–8 stop codons in all three reading frames flanked by 40 bp of microhomology to the disruption site, which was located within the first half of the open reading frame. Approximately 0.5–1 μg of each integration fragment was co-transformed with 500 ng of multiplex CRISPR plasmid targeting the desired genomic site. Positive integrants were identified by yeast colony PCR52, Sanger sequencing, and/or functional screening by LC–MS/MS.
Yeast strains were chemically transformed using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) as per the manufacturer’s instructions, with the following modifications. For competent cell preparation, individual colonies were inoculated into YPD media and grown overnight at 30 °C and 460 rpm. Saturated cultures (~14–18 h) were back-diluted between 1:10 and 1:50 in fresh YPD media and grown to exponential phase (~5–7 h). Cultures were pelleted by centrifugation at 500g for 4 min, washed twice with 50 mM Tris-HCl buffer (pH 8.5), and then resuspended in 20–50 μl of EZ2 solution per transformation. For transformation, competent cells were mixed with 250–1,000 ng of total DNA and 200–500 μl of EZ3 solution. Cell suspensions were incubated at 30 °C with slow rotation for 1–1.5 h. For plasmid transformations, the transformed yeast were directly plated onto YNB-DO agar plates. For CRISPRm genomic modifications, yeast suspensions were instead mixed with 1 ml YPD media, pelleted by centrifugation at 500g for 4 min, and then resuspended in 300–500 μl of fresh YPD medium. Suspensions were incubated at 30 °C with gentle rotation for 2–3 h to allow expression of geneticin resistance and then spread on YPD plates supplemented with 200–400 mg l−1 G418 (geneticin) sulfate. Plates were incubated at 30 °C for 72 h to allow sufficient colony formation before downstream applications.
Growth conditions for metabolite assays
Small-scale metabolite production assays were performed in YNB-SC or YNB-DO media supplemented with 2% dextrose and 5% glycerol (YNB-G) for optimal tropine production14 in at least three replicates. Our previous work showed that tropine biosynthesis is significantly enhanced by higher starting cell densities14. Therefore, yeast colonies were initially inoculated in triplicate into 1 ml YPD or YNB-DO and grown to saturation (~18–22 h) at 30 °C and 460 rpm, pelleted by centrifugation at 500g for 4 min and 3,000g for 1 min, resuspended in 1 ml of fresh selective or non-selective YNB-G media (for some experiments, additionally supplemented with 15 mg l−1 Fe2+ from iron (ii) sulfate and 50 mM 2-oxoglutarate56), and then 300 μl transferred into 2 ml deep-well 96-well plates sealed with AeraSeal gas-permeable film (Excel Scientific). Cultures were grown for 72–96 h at 25 °C, 460 rpm, and 80% relative humidity in a Lab-Therm LX-T shaker (Adolf Kuhner).
Growth conditions for time courses
To simulate high-density batch culture conditions, strains were inoculated in triplicate into 10 ml of YPD media or selective YNB-G media and grown overnight to saturation at 30 °C and 250 rpm. Saturated cultures were pelleted by centrifugation at 500g for 4 min and 3,000g for 1 min and then resuspended in 10 ml of fresh selective or non-selective YNB-G media supplemented with 50 mM 2-oxoglutarate and 15 mg l−1 Fe2+, and grown in 50-ml shake flasks with 10 ml starting volume in triplicates at 25 °C and 300 rpm for 120 h. Where indicated, fed-batch conditions were approximated by supplementing cultures after 72 h of growth with appropriate carbon sources and amino acids at 2% and 1× final concentrations, respectively. At appropriate time points, 250 μl samples were removed from cultures for analysis; 100 μl of culture was diluted 10× and used for optical density measurement at 600 nm on a Nanodrop 2000c spectrophotometer, and 150 μl of culture was used for metabolite quantification.
Analysis of metabolite production
Yeast cultures were pelleted by centrifugation at 3,500g for 5 min at 12 °C and 150 μl aliquots of supernatant were removed for analysis. Metabolites were analysed by LC–MS/MS using an Agilent 1260 Infinity Binary HPLC and an Agilent 6420 Triple Quadrupole mass spectrometer. Chromatography was performed using a Zorbax EclipsePlus C18 column (2.1 × 50 mm, 1.8 μm; Agilent Technologies) with 0.1% (v/v) formic acid in water as mobile phase solvent A and 0.1% (v/v) formic acid in acetonitrile as solvent B. The column was operated with a constant flow rate of 0.4 ml min−1 at 40 °C and a sample injection volume of 10 μl. Chromatographic separation was performed using the following gradient14: 0.00–0.75 min, 1% B; 0.75–1.33 min, 1–25% B; 1.33–2.70 min, 25–40% B; 2.70–3.70 min, 40–60% B; 3.70–3.71 min, 60–95% B; 3.71–4.33 min, 95% B; 4.33–4.34 min, 95–1% B; 4.34–5.00 min, equilibration with 1% B. For separation and detection of phenylpropanoid acyl donors (PLA, cinnamate and ferulate) and corresponding glucosides, the final equilibration step at 1% B was extended to 4.34–7.50 min. The LC eluent was directed to the MS from 0.01–5.00 min operating with electrospray ionization (ESI) in positive mode, source gas temperature 350 °C, gas flow rate 11 l min−1, and nebulizer pressure 40 psi. Data collection was performed using MassHunter Workstation LC/MS Data Acquisition software (Agilent). Metabolites were identified and quantified by integrated peak area in MassHunter Workstation Qualitative Analysis Navigator software (Agilent) using the mass fragment/transition parameters in Supplementary Table 4 and standard curves. Primary MRM transitions were identified by analysis of 0.1–1 mM aqueous standards using MassHunter Workstation Optimizer software (Agilent) and corroborated against published mass transitions if available, and/or against predicted transitions determined using the CFM-ID fragment prediction utility57 and the METLIN database58. As both PLA and its glucoside formed strong ammonium adducts, these metabolites were detected and quantified in positive mode using the corresponding [M + H + 17]+ ions, m/z 184 (PLA) and 346 (PLA glucoside) (Supplementary Table 4).
Individual colonies of yeast strains transformed with plasmids encoding biosynthetic enzymes fused to fluorescent protein reporters were inoculated into 1 ml selective or non-selective YNB-G media and grown overnight (~14–18 h) at 30 °C and 460 rpm. Overnight cultures were back-diluted between 1:2 and 1:4 into fresh YNB-G media and grown to exponential phase at 30 °C and 460 rpm for an additional 6–8 h to allow slow-maturing fluorescent proteins to fold before imaging.
Yeast vacuoles were co-imaged with fluorescent reporter-fused biosynthetic enzymes using the FM4-64 stain (Thermo Fisher) and pulse-chase fluorescence microscopy. FM4-64 is a red-fluorescent lipophilic styryl dye that intercalates into the yeast plasma membrane and is endocytosed during growth on rich media, accumulating in vacuolar membranes59. Transformed yeast colonies were inoculated into 1 ml selective or non-selective YNB-G and grown overnight (~14–18 h) at 30 °C and 460 rpm, then back-diluted between 1:10 and 1:3 into 1 ml of fresh YNB-G and grown for an additional 2–4 h until OD600 value of 0.5–0.8. Cultures were pelleted by centrifugation at 5,000g for 5 min, resuspended in 500 μl fresh YPD with 8 μM (5 ng μl−1) FM4-64, and incubated at 30 °C for 30 min with gentle rotation. Stained cells were pelleted by centrifugation at 3,000g for 5 min (pellets were visibly red), washed twice with 1 ml YPD, resuspended in 5 ml YPD, and then incubated at 30 °C and 460 rpm for 90–120 min to allow endocytosis and vacuolar accumulation of the dye. Cultures were pelleted by centrifugation at 500g for 4 min followed by 3,000g for 1 min, then resuspended in 250 μl of 40 mM MES buffer (pH 6.5) and imaged immediately.
For imaging, approximately 5–10 μl of cell suspension was spotted onto a glass microscope slide and covered with a glass coverslip (Thermo Fisher) and then imaged using an upright Zeiss AxioImager Epifluorescence/Widefield microscope with a × 64 oil immersion objective. Fluorescence excitation was performed using an EXFO X-Cite 120 illumination source and the following Semrock Brightline filter settings: GFP, 472/30 excitation and 520/35 emission; mCherry/DsRed/Cy3/TexasRed, 562/40 excitation and 624/40 emission. Emitted light was captured with a Zeiss Axiocam 503 mono camera and Zen Pro software, and subsequent image analysis was performed in ImageJ/Fiji (NIH). Images were converted to pseudocolor using the ‘merge channels’ and ‘split channels’ functions (Image→ Colour→Merge/Split Channels). For each sample, linear histogram stretching was applied across all images for a given channel to improve contrast.
To reduce the interference of light from other focal planes when imaging sub-cellular organelles, we performed 2D digital deconvolution analysis, a common computational technique used for removing out-of-focus light distortion from 2D images of 3D structures60. First, a theoretical point-spread function (PSF), which mathematically describes the diffraction of light from a point source in a specific imaging setup, was computed using the ‘Diffraction PSF 3D’ plugin for ImageJ (available from http://fiji.sc/Diffraction_PSF_3D) for the green and red channels using the following parameters: index of refraction of the media, 1.518 (lens oil); numerical aperture, 1.40; wavelength (nm), 520 (green) or 624 (red); longitudinal spherical aberration at max. aperture (nm), 0.00 (default); image pixel spacing (nm), 72; slice spacing (nm), 0; width (pixels), 240; height (pixels), 242; depth (slices), 1; normalization, sum of pixel values = 1. Next, green and red channel images were separately deconvolved against the corresponding PSFs using the ‘Parallel Spectral Deconvolution 2D’ plugin for ImageJ (available from http://fiji.sc/Parallel_Spectral_Deconvolution) with default settings and auto regularization.
Identification of HDH candidates
Tissue-specific abundances (fragments per kilobase of contig per million mapped reads, FPKM) and putative protein structural and functional annotations for each of 43,861 unique transcripts identified from the A. belladonna transcriptome were obtained from the MSU Medicinal Plant Genomics Resource30. Transcripts encoding hyoscyamine dehydrogenase candidates were identified based on clustering of tissue-specific expression profiles with those of the bait genes CYP80F1 (littorine mutase) and H6H (hyoscyamine 6β-hydroxylase/dioxygenase), which respectively precede and follow the dehydrogenase step in the TA biosynthetic pathway, using a custom R script which is described below.
First, the complete list of 43,861 transcripts was filtered for those annotated with any of the following oxidoreductase protein family (PFAM) IDs: PF00106, PF13561, PF08659, PF08240, PF00107, PF00248, PF00465, PF13685, PF13823, PF13602, PF16884 and PF00248; or any of the following functional annotation keywords: alcohol dehydrogenase, aldehyde reductase, short chain, aldo/keto. In addition, any transcripts with functional annotations containing the keywords putrescine, tropinone and tropine were included in the filter as positive control TA-associated genes to validate clustering with bait genes. Next, mean tissue-specific expression profiles were generated for the CYP80F1 and H6H bait genes. For each of the two bait genes, linear regression models were constructed to express the bait gene expression profile (in FPKM) as a linear function of each candidate gene profile and correlation P values were computed for each candidate. The candidates identified using each of the two bait genes were pooled and duplicates were removed. Combined P values for each candidate were computed as the sum of the log10(P values) of the correlations with each of the two bait genes. Transcripts matching known dehydrogenases in the TA biosynthetic pathway (that is, tropinone reductases I and II) were removed, and the remaining candidates were ranked by combined P value and by distance from bait genes via hierarchical clustering of tissue-specific expression profiles.
De novo transcriptome assembly
All data pre-processing, analysis, and de novo transcriptome assembly was performed on the Sherlock2.0 high-performance computing cluster (HPCC) at Stanford University. Paired-end raw RNA-seq reads corresponding to A. belladonna secondary roots (accession SRX060267, run SRR192881) and sterile seedling tissue (accession SRX060269, run SRR192882) were retrieved from the NCBI Sequence Read Archive (SRA) using the SRA Toolkit (NIH). The paired-end raw reads were analysed via FastQC (Babraham Bioinformatics) and then trimmed using the BBDuk.sh utility (Joint Genome Institute, Department of Energy) using the following parameters: k-mer trimming, right end only (‘ktrim=r’); k-mer length, 23 (‘k=23’); minimum k-mer for end-trimming, 11 (‘mink=11’), Hamming distance for k-mer matching, 1 (‘hdist=1’); trim paired reads to same length (‘tpe’), trim adapters using pair overlap detection (‘tbo’); quality trimming, both right and left ends (‘qtrim=rl’); quality cut-off, 5 (‘trimq=5’); minimum permissible read length after trimming, 25 (‘minlen=25’). Two independent de novo transcriptome assemblies were generated from the processed paired-end reads from secondary root (SRR192881) and seedling (SRR192882), respectively, using the Trinity software suite with default settings31,61.
Transcript functional annotation for each of the two assemblies (secondary root and seedling) was performed using the Trinotate package62. Following coding region prediction using the TransDecoder.LongOrfs and TransDecoder.Predict commands, annotations were generated using a BLASTp search against the UniProt/SwissProt databases and a protein homology search using HMMER. Complete ORF sequences for each of the candidate transcripts identified from co-expression analysis were generated by performing tBLASTn and tBLASTx searches against the Trinity transcriptome assemblies; hits with protein percent identity of at least 98%, accounting for sequencing errors, were assumed to be identical.
Identification of orthologues from transcriptome databases
Orthologues of A. belladonna UGT84A27 (UGT) were identified using tBLASTn searches of the transcriptomes of Brugmansia sanguinea and Datura metel in the 1000Plants (1KP) database63. This search yielded two unique, full-length amino acid sequences (that is, within 5% of the length of the query sequence) and with expectation value 0.0: scaffold-AIOU-2012986-Brugmansia_sanguinea (B. sanguinea, BsUGT) and scaffold-JNVS-2051323-Datura_metel (D. metel, DmUGT).
Orthologues of HDH were identified using tBLASTn searches of the transcriptomes of several Datura species in the Medicinal Plant RNA-seq database32. This search yielded two unique, full-length amino acid sequences (that is, within 5% of the length of the query sequence) and with expectation value 0.0: medp_datin_20101112|6354 (DiHDH) and medp_datst_20101112|10433 (DsHDH).
Coding sequences for all putative orthologues were optimized for yeast expression and then cloned into expression vectors as described in ‘Plasmid construction’.
Protein structural analysis and substrate docking
Homology models of AbUGT, AbHDH and AbLS were constructed using RaptorX with default modelling parameters64. For docking simulations, the binding of cosubstrates (UDP-glucose for AbUGT) or cofactors (NADPH for AbHDH) was first predicted based on structural alignment with the crystal structures of A. thaliana salicylate UDP-glucosyltransferase UGT74F2 with bound UDP (PDB: 5V2K) and Populus tremuloides sinapyl alcohol dehydrogenase with bound NADPH (PDB: 1YQD) respectively, as the binding pockets for these cosubstrates are tightly conserved. Geometry optimizations of substrate structures (PLA or hyoscyamine aldehyde) before docking simulations were conducted using the Gaussian 16 software package on the Stanford Sherlock2.0 HPCC (run parameters: DFT, B3LYP, LANL2DZ). The energy-minimized ligand structures were then docked into the corresponding cosubstrate/cofactor-bound homology models using the Maestro and Glide XP software packages (Schrodinger) with default parameters; for the docking of hyoscyamine aldehyde into AbHDH, a spatial constraint of maximum 6 Å separation between the aldehyde carbon and the catalytic Zn2+ was additionally imposed65. Enzyme structures and docking results were visualized using PyMOL software (Schrodinger).
Phylogenetic analysis of HDH orthologues
Phylogenetic tree construction was based on a BLASTp search using AbHDH as a query against the UniProt/SwissProt database (annotated sequences only). Sequences chosen for tree construction included the top 50 BLASTp hits based on E-value, as well as 10 additional hits selected from among the next 100 ranks. Phylogenetic relationships were derived via bootstrap neighbour-joining with n = 1,000 trials in ClustalX2 and the resulting tree was visualized with FigTree software.
Expression of littorine synthase HA-tagged variants in tobacco
Binary vector (pEAQ-HT-based) plasmids were transformed into A. tumefasciens (GV3101) using the freeze-thaw method66. Transformants were grown on LB-agar plates supplemented with 50 μg ml−1 kanamycin and 30 μg ml−1 gentamicin at 30 °C for 48 h. Colonies were inoculated into 5 ml liquid cultures of LB with 50 μg ml−1 kanamycin and 30 μg ml−1 gentamicin and grown for 18–24 h at 30 °C and 250 rpm in a shaking incubator. Saturated cultures were pelleted by centrifugation at 5,000g for 5 min. Pellets were resuspended in the same volume (~5 ml) of freshly prepared infiltration buffer (10 mM MES buffer, pH 5.6, 10 mM MgCl2, 150 μM acetosyringone), incubated at room temperature for 2–3 h with gentle rocking to prevent settling, and then diluted in infiltration buffer to OD600 of 0.8–1.0. N. benthamiana plants were grown for 4 weeks under a 16 h light/8 h dark cycle before infiltration. Approximately 1–2 ml of Agrobacterium cell suspension was infiltrated into the underside of each of the three largest leaves of each plant using a needleless 1 ml syringe. Leaves were harvested four days after infiltration, flash-frozen in liquid nitrogen, and stored at −80 °C for downstream processing. All infiltrations were performed in triplicate, in which one biological replicate comprised three infiltrated leaves from a single plant.
Deglycosylation of yeast- and tobacco-expressed littorine synthase
Removal of N- and O-linked glycosylation from littorine synthase in yeast and N. benthamiana crude cell lysate was performed using PNGase F and O-glycosidase (NEB), respectively, following the manufacturer’s protocols. In brief, approximately 30 μg of total protein containing LS in crude cell lysate was denatured in 1× glycoprotein denaturing buffer at 100 °C for 10 min, followed by immediate chilling on ice. Denatured lysates were deglycosylated using PNGase F or O-glycosidase as per manufacturer instructions at 37 °C for 1 h, then stored at −20 °C until analysis.
Analysis of protein expression by western blot
For immunoblot analysis of yeast-expressed proteins, strain CSY1294 was transformed with HA-tagged AbLS expression vectors as described in ‘Yeast transformations’. Three days after transformation, transformed colonies were inoculated into 2 ml YNB-DO media and grown overnight (~16–20 h) to stationary phase at 30 °C and 460 rpm. Cells were pelleted by centrifugation at 3,000g for 5 min, resuspended in 200 μl H2O, mixed with 200 μl of 0.2 M NaOH, and incubated at room temperature for 5 min to allow hydrolysis of cell wall glycoproteins67. Cells were re-pelleted at 3,000g for 5 min, resuspended in 75 μl H2O, mixed with 25 μl of 4× NuPAGE LDS sample buffer (Thermo Fisher), and then boiled at 95 °C for 3 min to lyse cells. Suspensions were pelleted by centrifugation at 16,000g for 5 min to remove insoluble debris and supernatants were transferred to pre-chilled tubes. Samples were stored at −20 °C until further analysis.
For analysis of tobacco-expressed proteins, all three infiltrated leaves from a single plant were ground together to a fine powder under liquid nitrogen and resuspended in 4–5 ml of 25 mM potassium phosphate buffer (pH 8.0) with HALT protease inhibitor cocktail (Thermo Fisher). Leaf homogenate slurries (final volume 7–8 ml) were incubated at 4 °C with gentle rotation for 45–60 min and then clarified by centrifugation at 9,000g for 10 min. Supernatant fractions were transferred to new tubes and re-clarified. Lysate protein concentrations were estimated using the Bio-RAD Protein Assay kit. Samples were stored at −80 °C until further analysis.
For analysis under reducing conditions, protein lysates were mixed with β-mercaptoethanol (final concentration 10%) and incubated at 70 °C for 10 min. Approximately 20–40 μg of total protein was loaded onto NuPAGE Bis-Tris 4–12% acrylamide gels (Thermo Fisher) with Precision Plus Dual Colour protein molecular mass marker (BioRad). Electrophoresis was conducted in 1× NuPAGE MOPS SDS running buffer at 150 V for 90 min. Transfer of protein to nitrocellulose membranes was performed at 15 V for 15 min using a Trans-Blot Semi-Dry apparatus (BioRad) and NuPAGE transfer buffer (Thermo Fisher) per manufacturer instructions. For reducing conditions, NuPAGE antioxidant (Thermo Fisher) was added to a final concentration of 1× to both the running buffer and transfer buffer. Membranes with transferred protein were washed for 5 min in Tris-buffered saline with Tween (TBS-T; 137 mM NaCl, 2.7 mM KCl, 19 mM Tris base, 0.1% Tween20, pH 7.4) and then blocked with 5% skim milk in TBS-T for 1 h at room temperature. Membranes were incubated overnight at 4 °C with a 1:1,500 dilution of chimaeric rabbit IgGκ anti-HA HRP-conjugated antibody (Absolute Antibody, 16.43/Ab00828-23.0) in TBS-T with 5% milk, washed three times for 5 min each with TBS-T, and then visualized using Western Pico PLUS HRP substrate (Thermo Fisher) and a G:BOX gel imager (Syngene).
Where indicated, the statistical significance of any differences in metabolite titer between conditions was verified using Student’s two-tailed t-test in Microsoft Excel Professional 2013. For yeast experiments, biological replicates are defined as independent cultures inoculated from separate yeast colonies or streaks and cultivated in separate containers. For tobacco experiments, one biological replicate is defined as all infiltrated leaves from a single plant.
All figures were prepared using GraphPad Prism 7, ImageJ, PyMOL, and Inkscape.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.